DNA sequencing by mass spectrometry

ABSTRACT

The invention describes a new method to sequence DNA. The improvements over the existing DNA sequencing technologies are high speed, high throughput, no electrophoresis and gel reading artifacts due to the complete absence of an electrophoretic step, and no costly reagents involving various substitutions with stable isotopes. The invention utilizes the Sanger sequencing strategy and assembles the sequence information by analysis of the nested fragments obtained by base-specific chain termination via their different molecular masses using mass spectrometry, as for example, MALDI or ES mass spectrometry. A further increase in throughput can be obtained by introducing mass-modifications in the oligonucleotide primer, chain-terminating nucleoside triphosphates and/or in the chain-elongating nucleoside triphosphates, as well as using integrated tag sequences which allow multiplexing by hybridization of tag specific probes with mass differentiated molecular weights.

This application is a divisional application of Ser. No. 08/178,216filed on Jan. 6, 1994, now U.S. Pat. No. 5,547,835 which is acontinuation in part of Ser. No. 08/001,323 filed Jan. 7, 1993, nowabandoned. The contents of all of the aforementioned application(s) arehereby incorporated by reference.

BACKGROUND OF THE INVENTION

Since the genetic information is represented by the sequence of the fourDNA building blocks deoxyadenosine-(dpA), deoxyguanosine-(dpG),deoxycytidine-(dpC) and deoxythymidine-5'-phosphate (dpT), DNAsequencing is one of the most fundamental technologies in molecularbiology and the life sciences in general. The ease and the rate by whichDNA sequences can be obtained greatly affects related technologies suchas development and production of new therapeutic agents and new anduseful varieties of plants and microorganisms via recombinant DNAtechnology. In particular, unraveling the DNA sequence helps inunderstanding human pathological conditions including genetic disorders,cancer and AIDS. In some cases, very subtle differences such as a onenucleotide deletion, addition or substitution can create serious, insome cases even fatal, consequences. Recently, DNA sequencing has becomethe core technology of the Human Genome Sequencing Project (e.g., J. E.Bishop and M. Waldholz, 1991, Genome: The Story of the Most AstonishingScientific Adventure of Our Time--The Attempt to Map All the Genes inthe Human Body, Simon & Schuster, New York). Knowledge of the completehuman genome DNA sequence will certainly help to understand, todiagnose, to prevent and to treat human diseases. To be able to tacklesuccessfully the determination of the approximately 3 billion base pairsof the human genome in a reasonable time frame and in an economical way,rapid, reliable, sensitive and inexpensive methods need to be developed,which also offer the possibility of automation. The present inventionprovides such a technology.

Recent reviews of today's methods together with future directions andtrends are given by Barrell (The FASEB Journal 5, 40-45(1991)), andTrainor (Anal. Chem. 62, 418-26 (1990)).

Currently, DNA sequencing is performed by either the chemicaldegradation method of Maxam and Gilbert (Methods in Enzymology 65,499-560 (1980)) or the enzymatic dideoxynucleotide termination method ofSanger et al. (Proc. Natl. Acad. Sci. USA 74, 5463-67 (1977)). In thechemical method, base specific modifications result in a base specificcleavage of the radioactive or fluorescently labeled DNA fragment. Withthe four separate base specific cleavage reactions, four sets of nestedfragments are produced which are separated according to length bypolyacrylamide gel electrophoresis (PAGE). After autoradiography, thesequence can be read directly since each band (fragment) in the geloriginates from a base specific cleavage event. Thus, the fragmentlengths in the four "ladders" directly translate into a specificposition in the DNA sequence.

In the enzymatic chain termination method, the four base specific setsof DNA fragments are formed by starting with a primer/template systemelongating the primer into the unknown DNA sequence area and therebycopying the template and synthesizing a complementary strand by DNApolymerases, such as Klenow fragment of E. coli DNA polymerase I, a DNApolymerase from Thermus aquaticus, Taq DNA polymerase, or a modified T7DNA polymerase, Sequenase (Tabor et al., Proc. Natl. Acad. Sci. USA 84,4767-4771 (1987)), in the presence of chain-terminating reagents. Here,the chain-terminating event is achieved by incorporating into the fourseparate reaction mixtures in addition to the four normaldeoxynucleoside triphosphates, dATP, dGTP, dTTP and dCTP, only one ofthe chain-terminating dideoxynucleoside triphosphates, ddATP, ddGTP,ddTTP or ddCTP, respectively, in a limiting small concentration. Thefour sets of resulting fragments produce, after electrophoresis, fourbase specific ladders from which the DNA sequence can be determined.

A recent modification of the Sanger sequencing strategy involves thedegradation of phosphorothioate-containing DNA fragments obtained byusing alpha-thio dNTP instead of the normally used ddNTPs during theprimer extension reaction mediated by DNA polymerase (Labeit et al., DNA5, 173-177 (1986); Amersham, PCT-Application GB86/00349; Eckstein etal., Nucleic Acids Res. 16,9947 (1988)). Here, the four sets ofbase-specific sequencing ladders are obtained by limited digestion withexonuclease III or snake venom phosphodiesterase, subsequent separationon PAGE and visualization by radioisotopic labeling of either the primeror one of the dNTPs. In a further modification, the base-specificcleavage is achieved by alkylating the sulphur atom in the modifiedphosphodiester bond followed by a heat treatment(Max-Planck-Gesellschaft, DE 3930312 A1). Both methods can be combinedwith the amplification of the DNA via the Polymerase Chain Reaction(PCR).

On the upfront end, the DNA to be sequenced has to be fragmented intosequencable pieces of currently not more than 500 to 1000 nucleotides.Starting from a genome, this is a multi-step process involving cloningand subcloning steps using different and appropriate cloning vectorssuch as YAC, cosmids, plasmids and M13 vectors (Sambrook et al.,Molecular Cloning: A Laboratory. Manual, Cold Spring Harbor LaboratoryPress, 1989). Finally, for Sanger sequencing, the fragments of about 500to 1000 base pairs are integrated into a specific restriction site ofthe replicative form I (RF I) of a derivative of the M13 bacteriophage(Vieria and Messing, Gene 19, 259 (1982)) and then the double-strandedform is transformed to the single-stranded circular form to serve as atemplate for the Sanger sequencing process having a binding site for auniversal primer obtained by chemical DNA synthesis (Sinha, Biernat,McManus and Koster, Nucleic Acids Res. 12, 4539-57 (1984); U.S. Pat. No.4,725,677 upstream of the restriction site into which the unknown DNAfragment has been inserted. Under specific conditions, unknown DNAsequences integrated into supercoiled double-stranded plasmid DNA can besequenced directly by the Sanger method (Chen and Seeburg, DNA 4,165-170 (1985)) and Lim et al., Gene Anal. Techn. 5, 32-39 (1988), and,with the Polymerase Chain Reaction (PCR) (PCR Protocols: A Guide toMethods and Applications. Innis et al., editors, Academic Press, SanDiego (1990)) cloning or subcloning steps could be omitted by directlysequencing off chromosomal DNA by first amplifying the DNA segment byPCR and then applying the Sanger sequencing method (Innis et al., Proc.Natl. Acad. Sci. USA 85, 9436-9440 (1988)). In this case, however, theDNA sequence in the interested region most be known at least to theextent to bind a sequencing primer.

In order to be able to read the sequence from PAGE, detectable labelshave to be used in either the primer (very often at the 5'-end) or inone of the deoxynucleoside triphosphates, dNTP. Using radioisotopes suchas ³² P, ³³ p, or ³⁵ S is still the most frequently used technique.After PAGE, the gels are exposed to X-ray films and silver grainexposure is analyzed. The use of radioisotopic labeling creates severalproblems. Most labels useful for autoradiographic detection ofsequencing fragements have relatively short half-lives which can limitthe useful time of the labels. The emission high energy beta radiation,particularly from ³² P, can lead to breakdown of the products viaradiolysis so that the sample should be used very quickly afterlabeling. In addition, high energy radiation can also cause adeterioration of band sharpness by scattering. Some of these problemscan be reduced by using the less energetic isotopes such as ³³ P or ³⁵ S(see, e.g., Ornstein et al., Biotechniques 3, 476 (1985)). Here,however, longer exposure times have to be tolerated. Above all, the useof radioisotopes poses significant health risks to the experimentalistand, in heavy sequencing projects, decontamination and handling theradioactive waste are other severe problems and burdens.

In response to the above mentioned problems related to the use ofradioactive labels, non-radioactive labeling techniques have beenexplored and, in recent years, integrated into partly automated DNAsequencing procedures. All these improvements utilize the Sangersequencing strategy. The fluorescent label can be tagged to the primer(Smith et al., Nature 321, 674-679 (1986) and EPO Pat. No. 87300998.9;Du Pont De Nemours EPO Application No. 0359225; Ansorge et al. J.Biochem. Biophys. Methods 13, 325-32 (1986)) or to the chain-terminatingdideoxynucloside triphosphates (Prober et al. Science 238, 336-41(1987); Applied Biosystems, PCT Application WO 91/05060). Based oneither labeling the primer or the ddNTP, systems have been developed byApplied Biosystems (Smith et at., Science 235, G89 (1987); U.S. Pat.Nos. 570,973 and 689,013), Du Pont De Nemours (Prober et al. Science238, 336-341 (1987); U.S. Pat. Nos. 881,372 and 57,566), Pharmacia-LKB(Ansorge et al. Nucleic Acids Res. 15, 4593-4602 (1987) and EMBL Pat.Application DE P3724442 and P3805808.1) and Hitachi (JP 1-90844 and DE4011991 A1). A somewhat similar approach was developed by Brumbaugh etal. (Proc. Natl. Sci. USA 85, 5610-14 (1988) and U.S. Pat. No.4,729,947). An improved method for the Du Pont system using twoelectrophoretic lanes with two different specific labels per lane isdescribed (PCT Application WO92/02635). A different approach usesfluorescently labeled avidin and biotin labeled primers. Here, thesequencing ladders ending with biotin are reacted during electrophoresiswith the labeled avidin which results in the detection of the individualsequencing bands (Brumbaugh et al, U.S. Pat. No. 594,676).

More recently even more sensitive non-radioactive labeling techniquesfor DNA using chemiluminescence triggerable and amplifyable by enzymeshave been developed (Beck, O'Keefe, Coull and Koster, Nucleic Acids Res.17, 5115-5123 (1989) and Beck and Koster, Anal. Chem. 62, 2258-2270(1990)). These labeling methods were combined with multiplex DNAsequencing (Church et al. Science 240, 185-188 (1988) to provide for astrategy aimed at high throughput DNA sequencing (Koster et al., NucleicAcids Res. Symposium Ser. No. 24, 318-321 (1991), University of Utah,PCT Application No. WO 90/15883); this strategy still suffers from thedisadvantage of being very laborious and difficult to automate.

In an attempt to simplify DNA sequencing, solid supports have beenintroduced. In most cases published so far, the template strand forsequencing (with or without PCR amplification) is immobilized on a solidsupport most frequently utilizing the strong biotin-avidin/streptavidininteraction (Orion-Yhtyma Oy, U.S. Pat. No. 277,643; M. Uhlen et al.Nucleic Acids Res. 16, 3025-38 (1988); Cemu Bioteknik, PCT ApplicationNo. WO 89/09282 and Medical Research Council, GB, PCT Application No. WO92/03575). The primer extension products synthesized on the immobilizedtemplate strand are purified of enzymes, other sequencing reagents andby-products by a washing step and then released under denaturingconditions by loosing the hydrogen bonds between the Watson-Crick basepairs and subjected to PAGE separation. In a different approach, theprimer extension products (not the template) from a DNA sequencingreaction are bound to a solid support via biotin/avidin (Du Pont DeNemours, PCT Application WO 91/11533). In contrast to the abovementioned methods, here, the interaction between biotin and avidin isovercome by employing denaturing conditions (formamide/EDTA) to releasethe primer extension products of the sequencing reaction from the solidsupport for PAGE separation. As solid supports, beads, (e.g., magneticbeads (Dynabeads) and Sepharose beads), filters, capillaries, plasticdipsticks (e.g., polystyrene strips) and microfiter wells are beingproposed.

All methods discussed so far have one central step in common:polyacrylamide gel electrophoresis (PAGE). In many instances, thisrepresents a major drawback and limitation for each of these methods.Preparing a homogeneous gel by polymerization, loading of the samples,the electrophoresis itself, detection of the sequence pattern (e.g., byautoradiography), removing the gel and cleaning the glass plates toprepare another gel are very laborious and time-consuming procedures.Moreover, the whole process is error-prone, difficult to automate, and,in order to improve reproducibility and reliability, highly trained andskilled personnel are required. In the case of radioactive labeling,autoradiography itself can consume from hours to days. In the case offluorescent labeling, at least the detection of the sequencing bands isbeing performed automatically when using the laser-scanning devicesintegrated into commercial available DNA sequencers. One problem relatedto the fluorescent labeling is the influence of the four differentbase-specific fluorescent tags on the mobility of the fragments duringelectrophoresis and a possible overlap in the spectral bandwidth of thefour specific dyes reducing the discriminating power between neighboringbands, hence, increasing the probability of sequence ambiguities.Artifacts are also produced by base-specific interactions with thepolyacrylamide gel matrix (Frank and Koster, Nucleic Acids Res. 6, 2069(1979)) and by the formation of secondary structures which result in"band compressions" and hence do not allow one to read the sequence.This problem has, in part, been overcome by using 7-deazadeoxyguanosinetriphosphates (Barr et al., Biotechniques 4, 428 (1986)). However, thereasons for some artifacts and conspicuous bands are still underinvestigation and need further improvement of the gel electrophoreticprocedure.

A recent innovation in electrophoresis is capillary zone electrophoresis(CZE) (Jorgenson et al., J. Chromatography 352, 337 (1986); Gesteland etal., Nucleic Acids Res. 18, 1415-1419 (1990)) which, compared to slabgel electrophoresis (PAGE), significantly increases the resolution ofthe separation, reduces the time for an electrophoretic run and allowsthe analysis of very small samples. Here, however, other problems arisedue to the miniaturization of the whole system such as wall effects andthe necessity of highly sensitive on-line detection methods. Compared toPAGE, another drawback is created by the fact that CZE is only a"one-lane" process, whereas in PAGE samples in multiple lanes can beelectrophoresed simultaneously.

Due to the severe limitations and problems related to having PAGE as anintegral and central part in the standard DNA sequencing protocol,several methods have been proposed to do DNA sequencing without anelectrophoretic step. One approach calls for hybridization orfragmentation sequencing (Bains, Biotechnology 10, 757-58 (1992) andMirzabekov et al., FEBS Letters 256, 118-122 (1989)) utilizing thespecific hybridization of known short oligonucleotides (e.g.,oetadeoxynucleotides which gives 65,536 different sequences) to acomplementary DNA sequence. Positive hybridization reveals a shortstretch of the unknown sequence. Repeating this process by performinghybridizations with all possible octadeoxynucleotides shouldtheoretically determine the sequence. In a completely differentapproach, rapid sequencing of DNA is done by unilaterally degrading onesingle, immobilized DNA fragment by an exonuclease in a moving flowstream and detecting the cleaved nucleotides by their specificfluorescent tag via laser excitation (Jett et al., J. BiomolecularStructure & Dynamics 7, 301-309, (1989); United States Department ofEnergy, PCT Application No. WO 89/03432). In another system proposed byHyman (Anal. Biochem. 174, 423-436 (1988)), the pyrophosphate generatedwhen the correct nucleotide is attached to the growing chain on aprimer-template system is used to determine the DNA sequence. Theenzymes used and the DNA are held in place by solid phases(DEAE-Sepharose and Sepharose) either by ionic interactions or bycovalent attachment. In a continuous flow-through system, the amount ofpyrophosphate is determined via bioluminescence (luciferase). Asynthesis approach to DNA sequencing is also used by Tsien et al. (PCTApplication No. WO 91/06678). Here, the incoming dNTP's are protected atthe 3'-end by various blocking groups such as acetyl or phosphate groupsand are removed before the next elongation step, which makes thisprocess very slow compared to standard sequencing methods. The templateDNA is immobilized on a polymer support. To detect incorporation, afluorescent or radioactive label is additionally incorporated into themodified dNTP's. The same patent application also describes an apparatusdesigned to automate the process.

Mass spectrometry, in general, provides a means of "weighing" individualmolecules by ionizing the molecules in vacuo and making them "fly" byvolatilization. Under the influence of combinations of electric andmagnetic fields, the ions follow trajectories depending on theirindividual mass (m) and charge (z). In the range of molecules with lowmolecular weight, mass spectrometry has long been part of the routinephysical-organic repertoire for analysis and characterization of organicmolecules by the determination of the mass of the parent molecular ion.In addition, by arranging collisions of this parent molecular ion withother particles (e.g., argon atoms), the molecular ion is fragmentedforming secondary ions by the so-called collision induced dissociation(CID). The fragmentation pattern/pathway very often allows thederivation of detailed structural information. Many applications of massspectrometric methods in the known in the art, particularly inbiosciences, and can be found summarized in Methods in Enzymology, Vol.193: "Mass Spectrometry" (J. A. McCloskey, editor), 1990, AcademicPress, New York.

Due to the apparent analytical advantages of mass spectrometry inproviding high detection sensitivity, accuracy of mass measurements,detailed structural information by CID in conjunction with an MS/MSconfiguration and speed, as well as on-line data transfer to a computer,there has been considerable interest in the use of mass spectrometry forthe structural analysis of nucleic acids. Recent reviews summarizingthis field include K. H. Schram, "Mass Spectrometry of Nucleic AcidComponents, Biomedical Applications of Mass Spectrometry" 34, 203-287(1990); and P. F. Crain, "Mass Spectrometric Techniques in Nucleic AcidResearch," Mass Spectrometry Reviews 9, 505-554 (1990). The biggesthurdle to applying mass spectrometry to nucleic acids is the difficultyof volatilizing these very polar biopolymers. Therefore, "sequencing"has been limited to low molecular weight synthetic oligonucleotides bydetermining the mass of the parent molecular ion and through this,confirming the already known sequence, or alternatively, confirming theknown sequence through the generation of secondary ions (fragment ions)via CID in an MS/MS configuration utilizing, in particular, for theionization and volatilization, the method of fast atomic bombardment(FAB mass spectrometry) or plasma desorption (PD mass spectrometry). Asan example, the application of FAB to the analysis of protected dimericblocks for chemical synthesis of oligodeoxynucleotides has beendescribed (Koster et al. Biomedical Environmental Mass Spectrometry 14,111-116 (1987)).

Two more recent ionization/desorption techniques areelectrospray/ionspray (ES) and matrix-assisted laserdesorption/ionization (MALDI). ES mass spectrometry has been introducedby Fenn et al. (J. Phys. Chem. 88, 4451-59 (1984); PCT Application No.WO 90/14148) and current applications are summarized in recent reviewarticles (R. D. Smith et al., Anal. Chem. 62, 882-89 (1990) and B.Ardrey, Electrospray Mass Spectrometry, Spectroscopy Europe, 4, 10-18(1992)). The molecular weights of the tetradecanucleotided(CATGCCATGGCATG) (SEQ ID NO:1) (Covey et al. "The Determination ofProtein, Oligonucleotide and Peptide Molecular Weights by Ionspray MassSpectrometry," Rapid Communications in Mass Spectrometry, 2, 249-256(1988)), of the 21-mer d(AAATTGTGCACATCCTGCAGC) (SEQ ID NO:2) andwithout giving details of that of a tRNA with 76 nucleotides (Methods inEnzymology, 193, "Mass Spectrometry" (McCloskey, editor), p. 425, 1990,Academic Press, New York) have been published. As a mass analyzer, aquadrupole is most frequently used. The determination of molecularweights in femtomole amounts of sample is very accurate due to thepresence of multiple ion peaks which all could be used for the masscalculation.

MALDI mass spectrometry, in contrast, can be particularly attractivewhen a time-of-flight (TOF) configuration is used as a mass analyzer.The MALDI-TOF mass spectrometry has been introduced by Hillenkamp et al.("Matrix Assisted UV-Laser Desorption/Ionization: A New Approach to MassSpectrometry of Large Biomolecules," Biological Mass Spectrometry(Burlingame and McCloskey, editors), Elsevier Science Publishers,Amsterdam, pp. 49-60, 1990.) Since, in most cases, no multiple molecularion peaks are produced with this technique, the mass spectra, inprinciple, look simpler compared to ES mass spectrometry. Although DNAmolecules up to a molecular weight of 410,000 daltons could be desorbedand volatilized (Williams et al., "Volatilization of High MolecularWeight DNA by Pulsed Laser Ablation of Frozen Aqueous Solutions,"Science, 246, 1585-87 (1989)), this technique has so far only been usedto determine the molecular weights of relatively small oligonucleotidesof known sequence, e.g., oligothymidylic acids up to 18 nucleotides(Huth-Fehre et al., "Matrix-Assisted Laser Desorption Mass Spectrometryof Oligodeoxythymidylic Acids," Rapid Communications in MassSpectrometry, 6, 209-13 (1992)) and a double-stranded DNA of 28 basepairs (Williams et al., "Time-of-Flight Mass Spectrometry of NucleicAcids by Laser Ablation and Ionization from a Frozen Aqueous Matrix,"Rapid Communications in Mass Spectrometry, 4, 348-351 (1990)). In onepublication (Huth-Fehre et al., 1992, supra), it was shown that amixture of all the oligothymidylic acids from n=12 to n=18 nucleotidescould be resolved.

In U.S. Pat. No. 5,064,754, RNA transcripts extended by DNA both ofwhich are complementary to the DNA to be sequenced are prepared byincorporating NTP's, dNTP's and, as terminating nucleotides, ddNTP'swhich are substituted at the 5'-position of the sugar moiety with one ora combination of the isotopes ¹² C, ¹³ C, ¹⁴ C, ¹ H, ² H, ³ H, ¹⁶ O, ¹⁷O and ¹⁸ O. The polynucleotides obtained are degraded to 3'-nucleotides,cleaved at the N-glycoside linkage and the isotopically labeled5'-functionality removed by periodate oxidation and the resultingformaldehyde species determined by mass spectrometry. A specificcombination of isotopes serves to discriminate base-specifically betweeninternal nucleotides originating from the incorporation of NTP's anddNTP's and terminal nucleotides caused by linking ddNTP's to the end ofthe polynucleotide chain. A series of RNA/DNA fragments is produced, andin one embodiment, separated by electrophoresis, and, with the aid ofthe so-called matrix method of analysis, the sequence is deduced.

In Japanese Patent No. 59-131909, an instrument is described whichdetects nucleic acid fragments separated either by electrophoresis,liquid chromatography or high speed gel filtration. Mass spectrometricdetection is achieved by incorporating into the nucleic acids atomswhich normally do not occur in DNA such as S, Br, I or Ag, Au, Pt, Os,Hg. The method, however, is not applied to sequencing of DNA using theSanger method. In particular, it does not propose a base-specificcorrelation of such elements to an individual ddNTP.

PCT Application No. WO 89/12694 (Brennan et al., Proc. SPIE-Int. Soc,Opt. Eng. 1206, (New Technol. Cytom. Mol. Biol.), pp. 60-77 (1990); andBrennan, U.S. Pat. No. 5,003,059) employs the Sanger methodology for DNAsequencing by using a combination of either the four stable isotopes ³²S, ³³ S, ³⁴ S, ³⁶ S or ³⁵ Cl, ³⁷ Cl, ⁷⁹ Br, ⁸¹ Br to specifically labelthe chain-terminating ddNTP's. The sulfur isotopes can be located eitherin the base or at the alpha-position of the triphosphate moiety whereasthe halogen isotopes are located either at the base or at the3'-position of the sugar ring. The sequencing reaction mixtures areseparated by an electrophoretic technique such as CZE, transferred to acombustion unit in which the sulfur isotopes of the incorporated ddNTP'sare transformed at about 900° C. in an oxygen atmosphere. The SO₂generated with masses of 64, 65, 66 or 68 is determined on-line by massspectrometry using, e.g., as mass analyzer, a quadrupole with a singleion-multiplier to detect the ion current.

A similar approach is proposed in U.S. Pat. No. 5,002,868 (Jacobson etal., Proc. SPIE-Int. Soc. Opt. Eng. 1435, (Opt. Methods UltrasensitiveDetect. Anal. Tech. Appl.), 26-35 (1991)) using Sanger sequencing withfour ddNTP's specifically substituted at the alpha-position of thetriphosphate moiety with one of the four stable sulfur isotopes asdescribed above and subsequent separation of the four sets of nestedsequences by tube gel electrophoresis. The only difference is the use ofresonance ionization spectroscopy (RIS) in conjunction with a magneticsector mass analyzer as disclosed in U.S. Pat. No. 4,442,354 to detectthe sulfur isotopes corresponding to the specific nucleotideterminators, and by this, allowing the assignment of the DNA sequence.

EPO Patent Applications No. 0360676 A1 and 0360677 A1 also describeSanger sequencing using stable isotope substitutions in the ddNTP's suchas D, ¹³ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ³² S, ³³ S, ³⁴ S, ³⁶ S, ¹⁹ F, ³⁵ Cl, ³⁷Cl, ⁷⁹ Br, ⁸¹ Br and ¹²⁷ I or function such as CF₃ or Si(CH₃)₃ at thebase, the sugar or the alpha position of the triphosphate moietyaccording to chemical functionality. The Sanger sequencing reactionmixtures are separated by tube gel electrophoresis. The effluent isconverted into an aerosol by the electrospray/thermospray nebulizermethod and then atomized and ionized by a hot plasma (7000° to 8000° K.)and analyzed by a simple mass analyzer. An instrument is proposed whichenables one to automate the analysis of the Sanger sequencing reactionmixture consisting of tube electrophoresis, a nebulizer and a massanalyzer.

The application of mass spectrometry to perform DNA sequencing by thehybridization/fragment method (see above) has been recently suggested(Bains, "DNA Sequencing by Mass Spectrometry: Outline of a PotentialFuture Application," Chimicaoggi 9, 13-16 (1991)).

SUMMARY OF THE INVENTION

The invention describes a new method to sequence DNA. The improvementsover the existing DNA sequencing technologies include high speed, highthroughput, no required electrophoresis (and, thus, no gel readingartifacts due to the complete absence of an electrophoretic step), andno costly reagents involving various substitutions with stable isotopes.The invention utilizes the Sanger sequencing strategy and assembles thesequence information by analysis of the nested fragments obtained bybase-specific chain termination via their different molecular massesusing mass spectrometry, for example, MALDI or ES mass spectrometry. Afurther increase in throughput can be obtained by introducing massmodifications in the oligonucleotide primer, the chain-terminatingnucleoside triphosphates and/or the chain-elongating nucleosidetriphosphates, as well as using integrated tag sequences which allowmultiplexing by hybridization of tag specific probes with massdifferentiated molecular weights.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a representation of a process to generate the samples to beanalyzed by mass spectrometry. This process entails insertion of a DNAfragment of unknown sequence into a cloning vector such as derivativesof M13, pUC or phagemids; transforming the double-stranded form into thesingle-stranded form; performing the four Sanger sequencing reactions;linking the base-specifically terminated nested fragment familytemporarily to a solid support; removing by a washing step allby-products; conditioning the nested DNA or RNA fragments by, forexample, cation-ion exchange or modification reagent and presenting theimmobilized nested fragments either directly to mass spectrometricanalysis or cleaving the purified fragment family off the support andevaporating the cleavage reagent.

FIG. 2A shows the Sanger sequencing products using ddTTP as terminatingdeoxynucleoside triphosphate of a hypothetical DNA fragment of 50nucleotides (SEQ ID NO:3) in length with approximately equally balancedbase composition. The molecular masses of the various chain terminatedfragments are given.

FIG. 2B shows an idealized mass spectrum of such a DNA fragment mixture.

FIGS. 3A and 3B show, in analogy to FIGS. 2A and 2B, data for the samemodel sequence (SEQ ID NO:3) with ddATP as chain terminator.

FIGS. 4A and 4B show data, analogous to FIGS. 2A and 2B when ddGTP isused as a chain terminator for the same model sequence (SEQ ID NO:3).

FIGS. 5A and 5B illustrate the results obtained where chain terminationis performed with ddCTP as a chain terminator, in a similar way as shownin FIGS. 2A and 2B for the same model sequence (SEQ ID NO:3).

FIG. 6 summarizes the results of FIGS. 2A to 5B, showing the correlationof molecular weights of the nested four fragment families to the DNAsequence (SEQ ID NO:3).

FIG. 7 illustrates the general structure of mass-modified sequencingnucleic acid primers or tag sequencing probes for either Sanger DNA orSanger RNA sequencing.

FIG. 8 shows the general structure for the mass-modified triphosphatesfor either Sanger DNA or Sanger RNA sequencing. General formulas of thechain-elongating and the chain-terminating nucleoside triphosphates aredemonstrated.

FIG. 9 outlines various linking chemistries (X) with either polyethyleneglycol or terminally monoalkylated polyethylene glycol (R) as anexample.

FIG. 10 illustrates similar linking chemistries as shown in FIG. 8 anddepicts various mass modifying moieties (R).

FIG. 11 outlines how multiplex mass spectrometric sequencing can workusing the mass-modified nucleic acid primer (UP).

FIG. 12 shows the process of multiplex mass spectrometric sequencingemploying mass-modified chain-elongating and/or terminating nucleosidetriphosphates.

FIG. 13 shows multiplex mass spectrometric sequencing by involving thehybridization of mass-modified tag sequence specific probes.

FIG. 14 shows a MALDI-TOF spectrum of a mixture of oligothymidylicacids, d(pT) 12-18.

FIG. 15 shows a superposition of MALDI-TOF spectra of the 50-merd(TAACGGTCATTACGGCCATTGACTGTAGGACCTGCATTACATGACTAGCT) (SEQ ID NO:3) (500fmol) and dT(pdT)₉₉ (500 fmol).

FIG. 16 shows the MALDI-TOF spectra of all 13 DNA sequences representingthe nested dT-terminated fragments of the Sanger DNA sequencingsimulation of FIG. 2, 500 fmol each.

FIG. 17 shows the superposition of the spectra of FIG. 16. The twopanels show two different scales and the spectra analyzed at that scale.

FIG. 18 shows the superimposed MALDI-TOF spectra from MALDI-MS analysisof mass-modified oligonucleotides as described in Example 21.

FIG. 19 illustrates various linking chemistries between the solidsupport (P) and the nucleic acid primer (NA) through a strongelectrostatic interaction.

FIG. 20 illustrates various linking chemistries between the solidsupport (P) and the nucleic acid primer (NA) through a charge transfercomplex of a charge transfer acceptor (A) and a charge transfer donor(D).

FIG. 21 illustrates various linking chemistries between the solidsupport (P) and the nucleic acid primer (NA) through a stable organicradical.

FIG. 22 illustrates a possible linking chemistry between the solidsupport (P) and the nucleic acid primer (NA) through Watson-Crick basepairing.

FIG. 23 illustrates linking the solid support (P) and the nucleic acidprimer (NA) through a photolytically cleavable bond.

DETAILED DESCRIPTION OF THE INVENTION

This invention describes an improved method of sequencing DNA. Inparticular, this invention employs mass spectrometry, such asmatrix-assisted laser desorption/ionization (MALDI) or electrospray (ES)mass spectrometry (MS), to analyze the Sanger sequencing reactionmixtures.

In Sanger sequencing, four families of chain-terminated fragments areobtained. The mass difference per nucleotide addition is 289.19 for dpC,313.21 for dpA, 329.21 for dpG and 304.2 for dpT, respectively.

In one embodiment, through the separate determination of the molecularweights of the four base-specifically terminated fragment families, theDNA sequence can be assigned via superposition (e.g., interpolation) ofthe molecular weight peaks of the four individual experiments. Inanother embodiment, the molecular weights of the four specificallyterminated fragment families can be determined simultaneously by MS,either by mixing the products of all four reactions run in at least twoseparate reaction vessels (i.e., all run separately, or two together, orthree together) or by running one reaction having all fourchain-terminating nucleotides (e.g., a reaction mixture comprising dTTP,ddTTP, dATP, ddATP, dCTP, ddCTP, dGTP, ddGTP) in one reaction vessel. Bysimultaneously analyzing all four base-specifically terminated reactionproducts, the molecular weight values have been, in effect,interpolated. Comparison of the mass difference measured betweenfragments with the known masses of each chain-terminating nucleotideallows the assignment of sequence to be carried out. In some instances,it may be desirable to mass modify, as discussed below, thechain-terminating nucleotides so as to expand the difference inmolecular weight between each nucleotide. It will be apparent to thoseskilled in the art when mass-modification of the chain-terminatingnucleotides is desirable and can depend, for instance, on the resolvingability of the particular spectrometer employed. By way of example, itmay be desirable to produce four chain-terminating nucleotides, ddTTP,ddCTP¹, ddATP² and ddGTP³ where ddCTP¹, ddATP² and ddGTP³ have each beenmass-modified so as to have molecular weights resolvable from oneanother by the particular spectrometer being used.

The terms chain-elongating nucleotides and chain-terminating nucleotidesare well known in the art. For DNA, chain-elongating nucleotides include2'-deoxyribonucleotides and chain-terminating nucleotides include2',3'-dideoxyribonucleotides. For RNA, chain-elongating nucleotidesinclude ribonucelotides and chain-terminating nucleotides include3'-deoxyribonucleotides. The term nucleotide is also well known in theart. For the purposes of this invention, nucleotides include nucleosidemono-, di-, and triphosphates. Nucleotides also include modifiednucleotides such as phosphorothioate nucleotides.

Since mass spectrometry is a serial method, in contrast to currentlyused slab gel electrophoresis which allows several samples to beprocessed in parallel, in another embodiment of this invention, afurther improvement can be achieved by multiplex mass spectrometric DNAsequencing to allow simultaneous sequencing of more than one DNA or RNAfragment. As described in more detail below, the range of about 300 massunits between one nucleotide addition can be utilized by employingeither mass-modified nucleic acid sequencing primers or chain-elongatingand/or terminating nucleoside triphosphates so as to shift the molecularweight of the base-specifically terminated fragments of a particular DNAor RNA species being sequenced in a predetermined manner. For the firsttime, several sequencing reactions can be mass spectrometricallyanalyzed in parallel. In yet another embodiment of this invention,multiplex mass spectrometric DNA sequencing can be performed by massmodifying the fragment families through specific oligonucleotides (tagprobes) which hybridize to specific tag sequences within each of thefragment families. In another embodiment, the tag probe can becovalently attached to the individual and specific tag sequence prior tomass spectrometry.

In one embodiment of the invention, the molecular weight values of atleast two base-specifically terminated fragments are determinedconcurrently using mass spectrometry. The molecular weight values ofpreferably at least five and more preferably at least tenbase-specifically terminated fragments are determined by massspectrometry. Also included in the invention are determinations of themolecular weight values of at least 20 base-specifically terminatedfragments and at least 30 base-specifically terminated fragments.Further, the nested base-specifically terminated fragments in a specificset can be purified of all reactants and by-products but are notseparated from one another. The entire set of nested base-specificallyterminated fragments is analyzed concurrently and the molecular weightvalues are determined. At least two base-specifically terminatedfragments are analyzed concurrently by mass spectrometry when thefragments are contained in the same sample.

In general, the overall mass spectrometric DNA sequencing process willstart with a library of small genomic fragments obtained after firstrandomly or specifically cutting the genomic DNA into large pieces whichthen, in several subcloning steps, are reduced in size and inserted intovectors like derivatives of M13 or pUC (e.g., M13mp18 or M13mp19) (seeFIG. 1). In a different approach, the fragments inserted in vectors,such as M13, are obtained via subcloning starting with a cDNA library.In yet another approach, the DNA fragments to be sequenced are generatedby the polymerase chain reaction (e.g., Higuchi et al., "A GeneralMethod of in vitro Preparation and Mutagenesis of DNA Fragments: Studyof Protein and DNA Interactions," Nucleic Acids Res., 16, 7351-67(1988)). As is known in the art, Sanger sequencing can start from onenucleic acid primer (UP) binding to the plus-strand or from anothernucleic acid primer binding to the opposite minus-strand. Thus, eitherthe complementary sequence of both strands of a given unknown DNAsequence can be obtained (providing for reduction of ambiguity in thesequence determination) or the length of the sequence informationobtainable from one clone can be extended by generating sequenceinformation from both ends of the unknown vector-inserted DNA fragment.

The nucleic acid primer carries, preferentially at the 5'-end, a linkingfunctionality, L, which can include a spacer of sufficient length andwhich can interact with a suitable functionality, L', on a solid supportto form a reversible linkage such as a photocleavable bond. Since eachof the four Sanger sequencing families starts with a nucleic acid primer(L-UP; FIG. 1) this fragment family can be bound to the solid support byreacting with functional groups, L', on the surface of a solid supportand then intensively washed to remove all buffer salts, triphosphates,enzymes, reaction by-products, etc. Furthermore, for mass spectrometricanalysis, it can be of importance at this stage to exchange the cationat the phosphate backbone of the DNA fragments in order to eliminatepeak broadening due to a heterogeneity in the cations bound pernucleotide unit. Since the L-L' linkage is only of a temporary naturewith the purpose to capture the nested Sanger DNA or RNA fragments toproperly condition them for mass spectrometric analysis, there aredifferent chemistries which can serve this purpose. In addition to theexamples given in which the nested fragments are coupled covalently tothe solid support, washed, and cleaved off the support for massspectrometric analysis, the temporary linkage can be such that it iscleaved under the conditions of mass spectrometry, i.e., aphotocleavable bond such as a charge transfer complex or a stableorganic radical. Furthermore, the linkage can be formed with L' being aquaternary ammonium group (some examples are given in FIG. 19). In thiscase, preferably, the surface of the solid support carries negativecharges which repel the negatively charged nucleic acid backbone andthus facilitates desorption. Desorption will take place either by theheat created by the laser pulse and/or, depending on L,' by specificabsorption of laser energy which is in resonance with the L' chromophore(see, e.g., examples given in FIG. 19). The functionalities, L and L,'can also form a charge transfer complex and thereby form the temporaryL-L' linkage. Various examples for appropriate functionalities witheither acceptor or donator properties are depicted without limitation inFIG. 20. Since in many cases the "charge-transfer band" can bedetermined by UV/vis spectrometry (see e.g. Organic Charge TransferComplexes by R. Foster, Academic Press, 1969), the laser energy can betuned to the corresponding energy of the charge-transfer wavelength and,thus, a specific desorption off the solid support can be initiated.Those skilled in the art will recognize that several combinations canserve this purpose and that the donor functionality can be either on thesolid support or coupled to the nested Sanger DNA/RNA fragments or viceversa.

In yet another approach, the temporary linkage L-L' can be generated byhomolytically forming relatively stable radicals as exemplified in FIG.21. In example 4 of FIG. 21, a combination of the approaches usingcharge-transfer complexes and stable organic radicals is shown. Here,the nested Sanger DNA/RNA fragments are captured via the formation of acharge transfer complex. Under the influence of the laser pulse,desorption (as discussed above) as well as ionization will take place atthe radical position. In the other examples of FIG. 21 under theinfluence of the laser pulse, the L-L' linkage will be cleaved and thenested Sanger DNA/RNA fragments desorbed and subsequently ionized at theradical position formed. Those skilled in the art will recognize thatother organic radicals can be selected and that, in relation to thedissociation energies needed to homolytically cleave the bond betweenthem, a corresponding laser wavelength can be selected (see e.g.Reactive Molecules by C. Wentrup, John Wiley & Sons, 1984). In yetanother approach, the nested Sanger DNA/RNA fragments are captured viaWatson-Crick base pairing to a solid support-bound oligonucleotidecomplementary to either the sequence of the nucleic acid primer or thetag oligonucleotide sequence (see FIG. 22). The duplex formed will becleaved under the influence of the laser pulse and desorption can beinitiated. The solid support-bound base sequence can be presentedthrough natural oligoribo- or oligodeoxyribonucleotide as well asanalogs (e.g. thio-modified phosphodiester or phosphotriester backbone)or employing oligonucleotide mimetics such as PNA analogs (see e.g.Nielsen et al., Science, 254, 1497 (1991)) which render the basesequence less susceptible to enzymatic degradation and hence increasesoverall stability of the solid support-bound capture base sequence. Withappropriate bonds, L-L', a cleavage can be obtained directly with alaser tuned to the energy necessary for bond cleavage. Thus, theimmobilized nested Sanger fragments can be directly ablated during massspectrometric analysis.

To increase mass spectrometric performance, it may be necessary tomodify the phosphodiester backbone prior to MS analysis. This can beaccomplished by, for example, using alpha-thio modified nucleotides forchain elongation and termination. With alkylating agents such asakyliodides, iodoacetamide, β-iodoethanol, 2,3-epoxy-1-propanol (seeFIG. 10), the monothio phosphodiester bonds of the nested Sangerfragments are transformed into phosphotriester bonds. Multiplexing bymass modification in this case is obtained by mass-modifying the nucleicacid primer (UP) or the nucleoside triphosphates at the sugar or thebase moiety. To those skilled in the art, other modifications of thenested Sanger fragments can be envisioned. In one embodiment of theinvention, the linking chemistry allows one to cleave off theso-purified nested DNA enzymatically, chemically or physically. By wayof example, the L-L' chemistry can be of a type of disulfide bond(chemically cleavable, for example, by mercaptoethanol ordithioerythrol), a biotin/streptavidin system, a heterobifunctionalderivative of a trityl ether group (Koster et al., "A VersatileAcid-Labile Linker for Modification of Synthetic Biomolecules,"Tetrahedron Letters 31, 7095 (1990)) which can be cleaved under mildlyacidic conditions, a levulinyl group cleavable under almost neutralconditions with a hydrazinium/acetate buffer, an arginine-arginine orlysine-lysine bond cleavable by an endopeptidase enzyme like trypsin ora pyrophosphate bond cleavable by a pyrophosphatase, a photocleavablebond which can be, for example, physically cleaved and the like (see,e.g., FIG. 23). Optionally, another cation exchange can be performedprior to mass spectrometric analysis. In the instance that anenzyme-cleavable bond is utilized to immobilize the nested fragments,the enzyme used to cleave the bond can serve as an internal massstandard during MS analysis.

The purification process and/or ion exchange process can be carried outby a number of other methods instead of, or in conjunction with,immobilization on a solid support. For example, the base-specificallyterminated products can be separated from the reactants by dialysis,filtration (including ultrafiltration), and chromatography. Likewise,these techniques can be used to exchange the cation of the phosphatebackbone with a counter-ion which reduces peak broadening.

The base-specifically terminated fragment families can be generated bystandard Sanger sequencing using the Large Klenow fragment of E. coliDNA polymerase I, by Sequenase, Taq DNA polymerase and other DNApolymerases suitable for this purpose, thus generating nested DNAfragments for the mass spectrometric analysis. It is, however, part ofthis invention that base-specifically terminated RNA transcripts of theDNA fragments to be sequenced can also be utilized for massspectrometric sequence determination. In this case, various RNApolymerases such as the SP6 or the T7 RNA polymerase can be used onappropriate vectors containing, for example, the SP6 or the T7 promoters(e.g. Axelrod et al, "Transcription from Bacteriophage T7 and SP6 RNAPolymerase Promoters in the Presence of 3'-Deoxyribonucleoside5'-triphosphate Chain Terminators," Biochemistry 24, 5716-23 (1985)). Inthis case, the unknown DNA sequence fragments are inserted downstreamfrom such promoters. Transcription can also be initiated by a nucleicacid primer (Pitulle et al., "Initiator Oligonucleotides for theCombination of Chemical and Enzymatic RNA Synthesis," Gene 112, 101-105(1992)) which carries, as one embodiment of this invention, appropriatelinking functionalities, L, which allow the immobilization of the nestedRNA fragments, as outlined above, prior to mass spectrometric analysisfor purification and/or appropriate modification and/or conditioning.

For this immobilization process of the DNA/RNA sequencing products formass spectrometric analysis, various solid supports can be used, e.g.,beads (silica gel, controlled pore glass, magnetic beads,Sephadex/Sepharose beads, cellulose beads, etc.), capillaries, glassfiber filters, glass surfaces, metal surfaces or plastic material.Examples of useful plastic materials include membranes in filter ormicrotiter plate formats, the latter allowing the automation of thepurification process by employing microtiter plates which, as oneembodiment of the invention, carry a permeable membrane in the bottom ofthe well functionalized with L'. Membranes can be based on polyethylene,polypropylene, polyamide, polyvinylidenedifluoride and the like.Examples of suitable metal surfaces include steel, gold, silver,aluminum, and copper. After purification, cation exchange, and/ormodification of the phosphodiester backbone of the L-L' bound nestedSanger fragments, they can be cleaved off the solid support chemically,enzymatically or physically. Also, the L-L' bound fragments can becleaved from the support when they are subjected to mass spectrometricanalysis by using appropriately chosen L-L' linkages and correspondinglaser energies/intensities as described above and in FIGS. 19-23.

The highly purified, four base-specifically terminated DNA or RNAfragment families are then analyzed with regard to their fragmentlengths via determination of their respective molecular weights by MALDIor ES mass spectrometry.

For ES, the samples, dissolved in water or in a volatile buffer, areinjected either continuously or discontinuously into an atmosphericpressure ionization interface (API) and then mass analyzed by aquadrupole. With the aid of a computer program, the molecular weightpeaks are searched for the known molecular weight of the nucleic acidprimer (UP) and determined which of the four chain-terminatingnucleotides has been added to the UP. This represents the firstnucleotide of the unknown sequence. Then, the second, the third, then^(th) extension product can be identified in a similar manner and, bythis, the nucleotide sequence is assigned. The generation of multipleion peaks which can be obtained using ES mass spectrometry can increasethe accuracy of the mass determination.

In MALDI mass spectrometry, various mass analyzers can be used, e.g.,magnetic sector/magnetic deflection instruments in single or triplequadrupole mode (MS/MS), Fourier transform and time-of-flight (TOF)configurations as is known in the art of mass spectrometry. FIGS. 2Athrough 6 are given as an example of the data obtainable when sequencinga hypothetical DNA fragment of 50 nucleotides in length (SEQ ID NO:3)and having a molecular weight of 15,344.02 daltons. The molecularweights calculated for the ddT (FIGS. 2A and 2B), ddA (FIGS. 3A and 3B),ddG (FIGS. 4A and 4B) and ddC (FIGS. 5A and 5B) terminated products aregiven (corresponding to fragments of SEQ ID NO:3) and the idealized fourMALDI-TOF mass spectra shown. All four spectra are superimposed, andfrom this, the DNA sequence can be generated. This is shown in thesummarizing FIG. 6, demonstrating how the molecular weights arecorrelated with the DNA sequence. MALDI-TOF spectra have been generatedfor the ddT terminated products (FIG. 16) corresponding to those shownin FIG. 2 and these spectra have been superimposed (FIG. 17). Thecorrelation of calculated molecular weights of the ddT fragments andtheir experimentally-verified weights are shown in Table 1. Likewise, ifall four chain-terminating reactions are combined and then analyzed bymass spectrometry, the molecular weight difference between two adjacentpeaks can be used to determine the sequence. For thedesorption/ionization process, numerous matrix/laser combinations can beused.

                  TABLE I                                                         ______________________________________                                        Correlation of calculated and experimentally verified molecular weights       of the 13 DNA fragments of FIGS. 2 and 16.                                    Fragment (n-mer)                                                                        calculated mass                                                                           experimental mass                                                                          difference                                 ______________________________________                                         7-mer    2104.45     2119.9       +15.4                                      10-mer    3011.04     3026.1       +15.1                                      11-mer    3315.24     3330.1       +14.9                                      19-mer    5771.82     5788.0       +16.2                                      20-mer    6076.02     6093.8       +17.8                                      24-mer    7311.82     7374.9       +63.1                                      26-mer    7945.22     7960.9       +15.7                                      33-mer    10112.63    10125.3      +12.7                                      37-mer    11348.43    11361.4      +13.0                                      38-mer    11652.62    11670.2      +17.6                                      42-mer    12872.42    12888.3      +15.9                                      46-mer    14108.22    14125.0      +16.8                                      50-mer    15344.02    15362.6      +18.6                                      ______________________________________                                    

In order to increase throughput to a level necessary for high volumegenomic and cDNA sequencing projects, a further embodiment of thepresent invention is to utilize multiplex mass spectrometry tosimultaneously determine more than one sequence. This can be achieved byseveral, albeit different, methodologies, the basic principle being themass modification of the nucleic acid primer (UP), the chain-elongatingand/or terminating nucleoside triphosphates, or by usingmass-differentiated tag probes hybridizable to specific tag sequences.The term "nucleic acid primer" as used herein encompasses primers forboth DNA and RNA Sanger sequencing.

By way of example, FIG. 7 presents a general formula of the nucleic acidprimer (UP) and the tag probes (TP). The mass modifying moiety can beattached, for instance, to either the 5'-end of the oligonucleotide(M¹), to the nucleobase (or bases) (M², M⁷), to the phosphate backbone(M³), and to the 2'-position of the nucleoside (nucleosides) (M⁴, M⁶)or/and to the terminal 3'-position (M⁵). Primer length can vary between1 and 50 nucleotides in length. For the priming of DNA Sangersequencing, the primer is preferentially in the range of about 15 to 30nucleotides in length. For artificially priming the transcription in aRNA polymerase-mediated Sanger sequencing reaction, the length of theprimer is preferentially in the range of about 2 to 6 nucleotides. If atag probe (TP) is to hybridize to the integrated tag sequence of afamily chain-terminated fragments, its preferential length is about 20nucleotides.

The table in FIG. 7 depicts some examples of mass-modified primer/tagprobe configurations for DNA, as well as RNA, Sanger sequencing. Thislist is, however, not meant to be limiting, since numerous othercombinations of mass-modifying functions and positions within theoligonucleotide molecule are possible and are deemed part of theinvention. The mass-modifying functionality can be, for example, ahalogen, an azido, or of the type, XR, wherein X is a linking group andR is a mass-modifying functionality. The mass-modifying functionalitycan thus be used to introduce defined mass increments into theoligonucleotide molecule.

In another embodiment, the nucleotides used for chain-elongation and/ortermination are mass-modified. Examples of such modified nucleotides areshown in FIG. 8. Here the mass-modifying moiety, M, can be attachedeither to the nucleobase, M² (in case of the c⁷ -deazanucleosides alsoto C-7, M⁷), to the triphosphate group at the alpha phosphate, M³, or tothe 2'-position of the sugar ring of the nucleoside triphosphate, M⁴ andM⁶. Furthermore, the mass-modifying functionality can be added so as toaffect chain termination, such as by attaching it to the 3'-position ofthe sugar ring in the nucleoside triphosphate, M⁵. The list in FIG. 8represents examples of possible configurations for generatingchain-terminating nucleoside triphosphates for RNA or DNA Sangersequencing. For those skilled in the art, however, it is clear that manyother combinations can serve the purpose of the invention equally well.In the same way, those skilled in the art will recognize thatchain-elongating nucleoside triphosphates can also be mass-modified in asimilar fashion with numerous variations and combinations infunctionality and attachment positions.

Without limiting the scope of the invention, FIG. 9 gives a moredetailed description of particular examples of how themass-modification, M, can be introduced for X in XR as well as usingoligo-/polyethylene glycol derivatives for R. The mass-modifyingincrement in this case is 44, i.e. five different mass-modified speciescan be generated by just changing m from 0 to 4 thus adding mass unitsof 45 (m=0), 89 (m=1), 133 (m=2), 177 (m=3) and 221 (m=4) to the nucleicacid primer (UP), the tag probe (TP) or the nucleoside triphosphatesrespectively. The oligo/polyethylene glycols can also be monoalkylatedby a lower alkyl such as methyl, ethyl, propyl, isopropyl, t-butyl andthe like. A selection of linking functionalities, X, are alsoillustrated. Other chemistries can be used in the mass-modifiedcompounds, as for example, those described recently in Oligonucleotidesand Analogues, A Practical Approach, F. Eckstein, editor, IRL Press,Oxford, 1991.

In yet another embodiment, various mass-modifying functionalities, R,other than oligo/polyethylene glycols, can be selected and attached viaappropriate linking chemistries, X. Without any limitation, someexamples are given in FIG. 10. A simple mass-modification can beachieved by substituting H for halogens like F, Cl, Br and/or I, orpseudohalogens such as SCN, NCS, or by using different alkyl, aryl oraralkyl moieties such as methyl, ethyl, propyl, isopropyl, t-butyl,hexyl, phenyl, substituted phenyl, benzyl, or functional groups such asCH₂ F, CHF₂, CF₃, Si(CH₃)₃, Si(CH₃)₂ (C₂ H₅), Si(CH₃)(C₂ H₅)₂, Si(C₂H₅)₃. Yet another mass-modification can be obtained by attaching homo-or heteropeptides through X to the UP, TP or nucleoside triphosphates.One example useful in generating mass-modified species with a massincrement of 57 is the attachment of oligoglycines, e.g.,mass-modifications of 74 (r=1, m=0), 131 (r=1, m=2), 188 (r=1, m=3), 245(r=1, m=4) are achieved. Simple oligoamides also can be used, e.g.,mass-modifications of 74 (r=1, m=0), 88 (r=2, m=0), 102 (r=3, m=0), 116(r=4, m=0), etc. are obtainable. For those skilled in the art, it willbe obvious that there are numerous possibilities in addition to thosegiven in FIG. 10 and the above mentioned reference (Oligonucleotides andAnalogues, F. Eckstein, 1991), for introducing, in a predeterminedmanner, many different mass-modifying functionalities to UP, TP andnucleoside triphosphates which are acceptable for DNA and RNA Sangersequencing.

As used herein, the superscript 0-i designates i+1 mass differentiatednucleotides, primers or tags. In some instances, the superscript 0(e.g., NTP⁰, UP⁰) can designate an unmodified species of a particularreactant, and the superscript i (e.g., NTP^(i), NTP¹, NTP², etc.) candesignate the i-th mass-modified species of that reactant. If, forexample, more than one species of nucleic acids (e.g., DNA clones) areto be concurrently sequenced by multiplex DNA sequencing, then i+1different mass-modified nucleic acid primers (UP⁰, UP¹, . . . UP^(i))can be used to distinguish each set of base-specifically terminatedfragments, wherein each species of mass-modified UP^(i) can bedistinguished by mass spectrometry from the rest.

As illustrative embodiments of this invention, three different basicprocesses for multiplex mass spectrometric DNA sequencing employing thedescribed mass-modified reagents are described below:

A) Multiplexing by the use of mass-modified nucleic acid primers (UP)for Sanger DNA or RNA sequencing (see for example FIG. 11);

B) Multiplexing by the use of mass-modified nucleoside triphosphates aschain elongators and/or chain terminators for Sanger DNA or RNAsequencing (see for example FIG. 12); and

C) Multiplexing by the use of tag probes which specifically hybridize totag sequences which are integrated into part of the four Sanger DNA/RNAbase-specifically terminated fragment families. Mass modification herecan be achieved as described for FIGS. 7, 9 and 10, or alternately, bydesigning different oligonucleotide sequences having the same ordifferent length with unmodified nucleotides which, in a predeterminedway, generate appropriately differentiated molecular weights (see forexample FIG. 13).

The process of multiplexing by mass-modified nucleic acid primers (UP)is illustrated by way of example in FIG. 11 for mass analyzing fourdifferent DNA clones simultaneously. The first reaction mixture isobtained by standard Sanger DNA sequencing having unknown DNA fragment 1(clone 1) integrated in an appropriate vector (e.g., M13mp18), employingan unmodified nucleic acid primer UP⁰, and a standard mixture of thefour modified deoxynucleoside triphosphates, dNTP⁰, and with 1/10th ofone of the four dideoxynucleoside triphosphates, ddNTP⁰. A secondreaction mixture for DNA fragment 2 (clone 2) is obtained by employing amass-modified nucleic acid primer UP¹ and, as before, the fourunmodified nucleoside triphosphates, dNTP⁰, containing in each separateSanger reaction 1/10th of the chain-terminating unmodifieddideoxynucleoside triphosphates ddNTP⁰. In the other two experiments,the four Sanger reactions have the following compositions: DNA fragment3 (clone 3), UP², dNTP⁰, ddNTP⁰ and DNA fragment 4 (clone 4), UP³,dNTP⁰, ddNTP⁰. For mass spectrometric DNA sequencing, allbase-specifically terminated reactions of the four clones are pooled andmass analyzed. The various mass peaks belonging to the fourdideoxy-terminated (e.g., ddT-terminated) fragment families are assignedto specifically elongated and ddT-terminated fragments by searching(such as by a computer program) for the known molecular ion peaks ofUP⁰, UP¹, UP² and UP³ extended by either one of the fourdideoxynucleoside triphosphates, UP⁰ -ddN⁰, UP¹ -ddN⁰, UP² -ddN⁰ and UP³-ddN⁰. In this way, the first nucleotides of the four unknown DNAsequences of clone 1 to 4 are determined. The process is repeated,having memorized the molecular masses of the four specific firstextension products, until the four sequences are assigned. Unambiguousmass/sequence assignments are possible even in the worst case scenarioin which the four mass-modified nucleic acid primers are extended by thesame dideoxynucleoside triphosphate, the extension products then being,for example, UP⁰ -ddT, UP¹ -ddT, UP² -ddT and UP³ -ddT, which differ bythe known mass increment differentiating the four nucleic acid primers.In another embodiment of this invention, an analogous technique isemployed using different vectors containing, for example, the SP6 and/orT7 promoter sequences, and performing transcription with the nucleicacid primers UP⁰, UP¹, UP² and UP³ and either an RNA polymerase (e.g.,SP6 or T7 RNA polymerase) with chain-elongating and terminatingunmodified nucleoside triphosphates NTP⁰ and 3'-dNTP⁰. Here, the DNAsequence is being determined by Sanger RNA sequencing.

FIG. 12 illustrates the process of multiplexing by mass-modifiedchain-elongating or/and terminating nucleoside triphosphates in whichthree different DNA fragments (3 clones) are mass analyzedsimultaneously. The first DNA Sanger sequencing reaction (DNA fragment1, clone 1) is the standard mixture employing unmodified nucleic acidprimer UP⁰, dNTP⁰ and in each of the four reactions one of the fourddNTP⁰. The second (DNA fragment 2, clone 2) and the third (DNA fragment3, clone 3) have the following contents: UP⁰, dNTP⁰, ddNTP¹ and UP⁰,dNTP⁰, ddNTP², respectively. In a variation of this process, anamplification of the mass increment in mass-modifying the extended DNAfragments can be achieved by either using an equally mass-modifieddeoxynucleoside triphosphate (i.e., dNTP¹, dNTP²) for chain elongationalone or in conjunction with the homologous equally mass-modifieddideoxynucleoside triphosphate. For the three clones depicted above, thecontents of the reaction mixtures can be as follows: either UP⁰ /dNTP⁰/ddNTP⁰, UP⁰ /dNTP ¹ /ddNTp⁰ and UP⁰ /dNTP² /ddNTP⁰ or Up⁰ /dNTp⁰/ddNTp⁰, Up⁰ /dNTp ¹ /ddNTP ¹ and UP⁰ /dNTP² /ddNTP². As describedabove, DNA sequencing can be performed by Sanger RNA sequencingemploying unmodified nucleic acid primers, UP⁰, and an appropriatemixture of chain-elongating and terminating nucleoside triphosphates.The mass-modification can be again either in the chain-terminatingnucleoside triphosphate alone or in conjunction with mass-modifiedchain-elongating nucleoside triphosphates. Multiplexing is achieved bypooling the three base-specifically terminated sequencing reactions(e.g., the ddTTP terminated products) and simultaneously analyzing thepooled products by mass spectrometry. Again, the first extensionproducts of the known nucleic acid primer sequence are assigned, e.g.,via a computer program. Mass/sequence assignments are possible even inthe worst case in which the nucleic acid primer is extended/terminatedby the same nucleotide, e.g., ddT, in all three clones. The followingconfigurations thus obtained can be well differentiated by theirdifferent mass-modifications: UP⁰ -ddT⁰, UP⁰ -ddT¹, UP⁰ -ddT².

In yet another embodiment of this invention, DNA sequencing by multiplexmass spectrometry can be achieved by cloning the DNA fragments to besequenced in "plex-vectors" containing vector specific "tag sequences"as described (Koster et al., "Oligonucleotide Synthesis and MultiplexDNA Sequencing Using Chemiluminescence Detection," Nucleic Acids Res.Symposium Ser. No. 24, 318-321 (1991)); then pooling clones fromdifferent plex-vectors for DNA preparation and the four separate Sangersequencing reactions using standard dNTP⁰ /ddNTP⁰ and nucleic acidprimer UP⁰ ; purifying the four multiplex fragment families via linkingto a solid support through the linking group, L, at the 5'-end of UP;washing out all by-products, and cleaving the purified multiplex DNAfragments off the support or using the L-L' bound nested Sangerfragments as such for mass spectrometric analysis as described above;performing de-multiplexing by one-by-one hybridization of specific "tagprobes"; and subsequently analyzing by mass spectrometry (see, forexample, FIG. 13). As a reference point, the four base-specificallyterminated multiplex DNA fragment families are run by the massspectrometer and all ddT⁰ -, ddA⁰ -, ddC⁰ - and ddG⁰ -terminatedmolecular ion peaks are respectively detected and memorized. Assignmentof, for example, ddT⁰ -terminated DNA fragments to a specific fragmentfamily is accomplished by another mass spectrometric analysis afterhybridization of the specific tag probe (TP) to the corresponding tagsequence contained in the sequence of this specific fragment family.Only those molecular ion peaks which are capable of hybridizing to thespecific tag probe are shifted to a higher molecular mass by the sameknown mass increment (e.g. of the tag probe). These shifted ion peaks,by virtue of all hybridizing to a specific tag probe, belong to the samefragment family. For a given fragment family, this is repeated for theremaining chain terminated fragment families with the same tag probe toassign the complete DNA sequence. This process is repeated i-1 timescorresponding to i clones multiplexed (the i-th clone is identified bydefault).

The differentiation of the tag probes for the different multiplexedclones can be obtained just by the DNA sequence and its ability toWatson-Crick base pair to the tag sequence. It is well known in the arthow to calculate stringency conditions to provide for specifichybridization of a given tag probe with a given tag sequence (see, forexample, Molecular Cloning: A laboratory manual 2ed by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: New York, 1989,Chapter 11). Furthermore, differentiation can be obtained by designingthe tag sequence for each plex-vector to have a sufficient massdifference so as to be unique just by changing the length or basecomposition or by mass-modifications according to FIGS. 7, 9 and 10. Inorder to keep the duplex between the tag sequence and the tag probeintact during mass spectrometric analysis, it is another embodiment ofthe invention to provide for a covalent attachment mediated by, forexample, photoreactive groups such as psoralen and ellipticine and byother methods known to those skilled in the art (see, for example,Heleet al., Nature 344, 358 (1990) and Thuong et al. "OligonucleotidesAttached to Intercalators, Photoreactive and Cleavage Agents" in F.Eckstein, Oligonucleotides and Analogues: A Practical Approach, IRLPress, Oxford 1991, 283-306).

The DNA sequence is unraveled again by searching for the lowestmolecular weight molecular ion peak corresponding to the known UP⁰ -tagsequence/tag probe molecular weight plus the first extension product,e.g., ddT⁰, then the second, the third, etc.

In a combination of the latter approach with the previously describedmultiplexing processes, a further increase in multiplexing can beachieved by using, in addition to the tag probe/tag sequenceinteraction, mass-modified nucleic acid primers (FIG. 7) and/ormass-modified deoxynucleoside, dNTP^(0-i), and/or dideoxynucleosidetriphosphates, ddNTP^(0-i). Those skilled in the art will realize thatthe tag sequence/tag probe multiplexing approach is not limited toSanger DNA sequencing generating nested DNA fragments with DNApolymerases. The DNA sequence can also be determined by transcribing theunknown DNA sequence from appropriate promoter-containing vectors (seeabove) with various RNA polymerases and mixtures of NTP^(0-i)/3'-dNTP^(0-i), thus generating nested RNA fragments.

In yet another embodiment of this invention, the mass-modifyingfunctionality can be introduced by a two or multiple step process. Inthis case, the nucleic acid primer, the chain-elongating or terminatingnucleoside triphosphates and/or the tag probes are, in a first step,modified by a precursor functionality such as azido, --N₃, or modifiedwith a functional group in which the R in XR is H (FIGS. 7, 9) thusproviding temporary functions, e.g., but not limited to --OH, --NH₂,--NHR, --SH, --NCS, --OCO(CH₂)_(r) COOH (r=1-20), --NHCO(CH₂)_(r) COOH(r=1-20), --OSO₂ OH, --OCO(CH₂)_(r) I (r=1-20), --OP(O--Alkyl)N(Alkyl)₂.These less bulky functionalities result in better substrate propertiesfor the enzymatic DNA or RNA synthesis reactions of the DNA sequencingprocess. The appropriate mass-modifying functionality is then introducedafter the generation of the nested base-specifically terminated DNA orRNA fragments prior to mass spectrometry. Several examples of compoundswhich can serve as mass-modifying functionalities are depicted in FIGS.9 and 10 without limiting the scope of this invention.

Another aspect of this invention concems kits for sequencing nucleicacids by mass spectrometry which include combinations of theabove-described sequencing reactants. For instance, in one embodiment,the kit comprises reactants for multiplex mass spectrometric sequencingof several different species of nucleic acid. The kit can include asolid support having a linking functionality (L¹) for immobilization ofthe base-specifically terminated products; at least one nucleic acidprimer having a linking group (L) for reversibly and temporarily linkingthe primer and solid support through, for example, a photocleavablebond; a set of chain-elongating nucleotides (e.g., dATP, dCTP, dGTP anddTTP, or ATP, CTP, GTP and UTP); a set of chain-terminating nucleotides(such as 2',3'-dideoxynucleotides for DNA synthesis or3'-deoxynucleotides for RNA synthesis); and an appropriate polymerasefor synthesizing complementary nucleotides. Primers and/or terminatingnucleotides can be mass-modified so that the base-specificallyterminated fragments generated from one of the species of nucleic acidsto be sequenced can be distinguished by mass spectrometry from all ofthe others. Alternative to the use of mass-modified synthesis reactants,a set of tag probes (as described above) can be included in the kit. Thekit can also include appropriate buffers as well as instructions forperforming multiplex mass spectrometry to concurrently sequence multiplespecies of nucleic acids.

In another embodiment, a nucleic acid sequencing kit can comprise asolid support as described above, a primer for initiating synthesis ofcomplementary nucleic acid fragments, a set of chain-elongatingnucleotides and an appropriate polymerase. The mass-modifiedchain-terminating nucleotides are selected so that the addition of oneof the chain terminators to a growing complementary nucleic acid can bedistinguished by mass spectrometry.

EXAMPLE 1

Immobilization of primer-extension products of Sanger DNA sequencingreaction for mass spectrometric analysis via disulfide bonds.

As a solid support, Sequelon membranes (Millipore Corp., Bedford, Mass.)with phenyl isothiocyanate groups are used as a starting material. Themembrane disks, with a diameter of 8 mm, are wetted with a solution ofN-methylmorpholine/water/2-propanol (NMM solution) (2/49/49 v/v/v), theexcess liquid removed with filter paper and placed on a piece of plasticfilm or aluminum foil located on a heating block set to 55° C. Asolution of 1 mM 2-mercaptoethylamine (cysteamine) or2,2'-dithio-bis(ethylamine) (cystamine) orS-(2-thiopyridyl)-2-thio-ethylamine (10 ul, 10 nmol) in NMM is added perdisk and heated at 55° C. After 15 min, 10 ul of NMM solution are addedper disk and heated for another 5 min. Excess of isothiocyanate groupsmay be removed by treatment with 10 ul of a 10 mM solution of glycine inNMM solution. For cystamine, the disks are treated with 10 ul of asolution of 1M aqueous dithiothreitol (DTT)/2-propanol (1:1 v/v) for 15min at room temperature. Then, the disks are thoroughly washed in afiltration manifold with 5 aliquots of 1 ml each of the NMM solution,then with 5 aliquots of 1 ml acetonitrile/water (1/1 v/v) andsubsequently dried. If not used immediately the disks are stored withfree thiol groups in a solution of 1M aqueous dithiothreitol/2-propanol(1:1 v/v) and, before use, DTT is removed by three washings with 1 mleach of the NMM solution. The primer oligonucleotides with 5'-SHfunctionality can be prepared by various methods (e.g., B. C. F Chu etal., Nucleic Acids Res. 14, 5591-5603 (1986), Sproat et al., NucleicAcids Res. 15, 4837-48 (1987) and Oligonucleotides and Analogues: APractical Approach (F. Eckstein, editor), IRL Press Oxford, 1991).Sequencing reactions according to the Sanger protocol are performed in astandard way (e.g., H. Swerdlow et al., Nucleic Acids Res. 18, 1415-19(1990)). In the presence of about 7-10 mM DTT the free 5'-thiol primercan be used; in other cases, the SH functionality can be protected,e.g., by a trityl group during the Sanger sequencing reactions andremoved prior to anchoring to the support in the following way. The foursequencing reactions (150 ul each in an Eppendorf tube) are terminatedby a 10 min incubation at 70° C. to denature the DNA polymerase (such asKlenow fragment, Sequenase) and the reaction mixtures are ethanolprecipitated. The supernatants are removed and the pellets vortexed with25 ul of an 1M aqueous silver nitrate solution, and after one hour atroom temperature, 50 ul of an 1M aqueous solution of DTT is added andmixed by vortexing. After 15 min, the mixtures are centrifuged and thepellets are washed twice with 100 ul ethylacetate by vortexing andcentrifugation to remove excess DTT. The primer extension products withfree 5'-thiol group are now coupled to the thiolated membrane supportsunder mild oxidizing conditions. In general, it is sufficient to add the5'-thiolated primer extension products dissolved in 10 ul 10 mMde-aerated triethylammonium acetate buffer (TEAA) pH 7.2 to thethiolated membrane supports. Coupling is achieved by drying the samplesonto the membrane disks with a cold fan. This process can be repeated bywetting the membrane with 10 ul of 10 mM TEAA buffer pH 7.2 and dryingas before. When using the 2-thiopyridyl derivatized compounds, anchoringcan be monitored by the release of pyridine-2-thionespectrophotometrically at 343 nm.

In another variation of this approach, the oligonucleotide primer isfunctionalized with an amino group at the 5'-end which is introduced bystandard procedures during automated DNA synthesis. After primerextension, during the Sanger sequencing process, the primary amino groupis reacted with 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimideester (SPDP) and subsequently coupled to the thiolated supports andmonitored by the release of pyridyl-2-thione as described above. Afterdenaturation of DNA polymerase and ethanol precipitation of thesequencing products, the supernalants are removed and the pelletsdissolved in 10 ul 10 mM TEAA buffer pH 7.2 and 10 ul of a 2 mM solutionof SPDP in 10 mM TEAA are added. The reaction mixture is vortexed andincubated for 30 min at 25° C. Excess SPDP is then removed by threeextractions (vortexing, centrifugation) with 50 ul each of ethanol andthe resulting pellets are dissolved in 10 ul 10 mM TEAA buffer pH 7.2and coupled to the thiolated supports (see above).

The primer-extension products are purified by washing the membrane disksthree times each with 100 ul NMM solution and three times with 100 uleach of 10 mM TEAA buffer pH 7.2. The purified primer-extension productsare released by three successive treatments with 10 ul of 10 mM2-mercaptoethanol in 10 mM TEAA buffer pH 7.2, lyophilized and analyzedby either ES or MALDI mass spectrometry.

This procedure can also be used for the mass-modified nucleic acidprimers UP^(0-i) in an analogous and appropriate way, taking intoaccount the chemical properties of the mass-modifying functionalities.

EXAMPLE 2

Immobilization of primer-extension products of Sanger DNA sequencingreaction for mass spectrometric analysis via the levulinyl group

5-Aminolevulinic acid is protected at the primary amino group with theFmoc group using 9-fluorenylmethyl N-succinimidyl carbonate and is thentransformed into the N-hydroxysuccinimide ester (NHS ester) usingN-hydroxysuccinimide and dicyclohexyl carbodiimide under standardconditions. For the Sanger sequencing reactions, nucleic acid primers,UP^(0-i), are used which are functionalized with a primary amino groupat the 5'-end introduced by standard procedures during automated DNAsynthesis with aminolinker phosphoamidites as the final synthetic step.Sanger sequencing is performed under standard conditions (see above).The four reaction mixtures (150 ul each in an Eppendorf tube) are heatedto 70° C. for 10 min to inactivate the DNA polymerase, ethanolprecipitated, centrifuged and resuspended in 10 ul of 10 mM TEAA bufferpH 7.2. 10 ul of a 2 mM solution of the Fmoc-5-aminolevulinyl-NHS esterin 10 mM TEAA buffer is added, vortexed and incubated at 25° C. for 30min. The excess of the reagent is removed by ethanol precipitation andcentrifugation. The Fmoc group is cleaved off by resuspending thepellets in 10 ul of a solution of 20% piperidine inN,N-dimethylformamide/water (1:1 v/v). After 15 min at 25° C.,piperidine is thoroughly removed by three precipitations/centrifugationswith 100 ul each of ethanol, the pellets are resuspended in 10 ul of asolution of N-methylmorpholine, 2-propanol and water (2/10/88 v/v/v) andare coupled to the solid support carrying an isothiocyanate group. Inthe case of the DITC-Sequelon membrane (Millipore Corp., Bedford,Mass.), the membranes are prepared as described in EXAMPLE 1 andcoupling is achieved on a heating block at 55° C. as described above.RNA extension products are immobilized in an analogous way. Theprocedure can be applied to other solid supports with isothiocyanategroups in a similar manner.

The immobilized primer-extension products are extensively washed threetimes with 100 ul each of NMM solution and three times with 100 ul 10 mMTEAA buffer pH 7.2. The purified primer-extension products are releasedby three successive treatments with 10 ul of 100 mM hydrazinium acetatebuffer pH 6.5, lyophilized and analyzed by either ES or MALDI massspectrometry.

EXAMPLE 3

Immobilization of primer-extension products of Sanger DNA sequencingreaction for mass spectrometric analysis via a trypsin sensitive linkage

Sequelon DITC membrane disks of 8 mm diameter (Millipore Corp., Bedford,Mass.) are wetted with 10 ul of NMM solution(N-methylmorpholine/propanaol-2/water; 2/49/49 v/v/v) and a linker armintroduced by reaction with 10 ul of a 10 mM solution of1,6-diaminohexane in NMM. The excess diamine is removed by three washingsteps with 100 ul of NMM solution. Using standard peptide synthesisprotocols, two L-lysine residues are attached by two successivecondensations with N-Fmoc-N-tBoc-L-lysine pentafluorophenylester, theterminal Fmoc group is removed with piperidine in NMM and the freeα-amino group coupled to 1,4-phenylene diisothioeyanate (DITC). ExcessDITC is removed by three washing steps with 100 ul 2-propanol and theN-tBoc groups removed with trifluoroacetic acid according to standardpeptide synthesis procedures. The nucleic acid primer-extension productsare prepared from oligonucleotides which carry a primary amino group atthe 5'-terminus. The four Sanger DNA sequencing reaction mixtures (150ul each in Eppendorf tubes) are heated for 10 min at 70° C. toinactivate the DNA polymerase, ethanol precipitated, and the pelletsresuspended in 10 ul of a solution of N-methylmorpholine, 2-propanol andwater (2/10/88 v/v/v). This solution is transferred to the Lys-Lys-DITCmembrane disks and coupled on a heating block set at 55° C. Afterdrying, 10 ul of NMM solution is added and the drying process repeated.

The immobilized primer-extension products are extensively washed threetimes with 100 ul each of NMM solution and three times with 100 ul eachof 10 mM TEAA buffer pH 7.2. For mass spectrometric analysis, the bondbetween the primer-extension products and the solid support is cleavedby treatment with trypsin under standard conditions and the releasedproducts analyzed by either ES or MALDI mass spectrometry with trypsinserving as an internal mass standard.

EXAMPLE 4

Immobilization of primer-extension products of Sanger DNA sequencingreaction for mass spectrometric analysis via pyrophosphate linkage

The DITC Sequelon membrane (disks of 8 mm diameter) are prepared asdescribed in EXAMPLE 3 and 10 ul of a 10 mM solution of 3-aminopyridineadenine dinucleotide (APAD) (Sigma) in NMM solution added. The excessAPAD is removed by a 10 ul wash of NMM solution and the disks aretreated with 10 ul of 10 mM sodium periodate in NMM solution (15 min,25° C.). Excess periodate is removed and the primer-extension productsof the four Sanger DNA sequencing reactions (150 ul each in Eppendorftubes) employing nucleic acid primers with a primary amino group at the5'-end are ethanol precipitated, dissolved in 10 ul of a solution ofN-methylmorpholine/2-propanol/water (2/10/88 v/v/v) and coupled to the2'3'-dialdehydo groups of the immobilized AND analog.

The primer-extension products are extensively washed with the NMMsolution (3 times with 100 ul each) and 10 mM TEAA buffer pH 7.2 (3times with 100 ul each) and the purified primer-extension products arereleased by treatment with either NADase or pyrophosphatase in 10 mMTEAA buffer at pH 7.2 at 37° C. for 15 min, lyophilized and analyzed byeither ES or MALDI mass spectrometry, the enzymes serving as internalmass standards.

EXAMPLE 5

Synthesis of nucleic acid primers mass-modified by glycine residues atthe 5'-position of the sugar moiety of the terminal nucleoside

Oligonucleotides are synthesized by standard automated DNA synthesisusing β-cyanoethylphosphoamidites (H. Koster et al., Nucleic Acids Res.12, 4539 (1984)) and a 5'-amino group is introduced at the end of solidphase DNA synthesis (e.g. Agrawal et al., Nucleic Acids Res. 14, 6227-45(1986) or Sproat et al., Nucleic Acids Res. 15, 6181-96 (1987)). Thetotal mount of an oligonucleotide synthesis, starting with 0.25 umolCPG-bound nucleoside, is deprotected with concentrated aqueous ammonia,purified via OligoPAK™ Cartridges (Millipore Corp., Bedford, Mass.) andlyophilized. This material with a 5'-terminal amino group is dissolvedin 100 ul absolute N,N-dimethylformamide (DMF) and condensed with 10μmole N-Fmoc-glycine pentafluorophenyl ester for 60 min at 25° C. Afterethanol precipitation and centrifugation, the Fmoc group is cleaved offby a 10 min treatment with 100 ul of a solution of 20% piperidine inN,N-dimethylformamide. Excess piperidine, DMF and the cleavage productfrom the Fmoc group are removed by ethanol precipitation and theprecipitate lyophilized from 10 mM TEAA buffer pH 7.2. This material isnow either used as primer for the Sanger DNA sequencing reactions or oneor more glycine residues (or other suitable protected amino acid activeesters) are added to create a series of mass-modified primeroligonucleotides suitable for Sanger DNA or RNA sequencing.Immobilization of these mass-modified nucleic acid primers UP^(0-i)after primer-extension during the sequencing process can be achieved asdescribed, e.g., in EXAMPLES 1 to 4.

EXAMPLE 6

Synthesis of nucleic acid primers mass-modified at C-5 of theheterocyclic base of a pyrimidine nucleoside with glycine residues

Starting material was 5-(3-aminopropynyl-1)-3'5'-di-p-tolyldeoxyuridineprepared and 3'5'-de-O-acylated according to literature procedures(Haralambidis et al., Nucleic Acids Res. 15, 4857-76 (1987)). 0.281 g(1.0 mmole) 5-(3-aminopropynyl-1)-2'-deoxyuridine were reacted with0.927 g (2.0 mmole) N-Fmoc-glycine pentafluorophenylester in 5 mlabsolute N,N-dimethylformamide in the presence of 0.129 g (1 mmole; 174ul) N,N-diisopropylethylamine for 60 min at room temperature. Solventswere removed by rotary evaporation and the product was purified bysilica gel chromatography (Kieselgel 60, Merck; column: 2.5×50 cm,elution with chloroform/methanol mixtures). Yield was 0.44 g (0.78mmole, 78%). In order to add another glycine residue, the Fmoc group isremoved with a 20 min treatment with 20% solution of piperidine in DMF,evaporated in vacuo and the remaining solid material extracted threetimes with 20 ml ethylacetate. After having removed the remainingethylacetate, N-Fmoc-glycine pentafluorophenylester is coupled asdescribed above. 5-(3-(N-Fmoc-glycyl)-aminopropynyl-1)-2'-deoxyuridineis transformed into the 5'-O-dimethoxytritylatednucleoside-3'-O-β-cyanoethyl-N,N-diisopropylphosphoamidite andincorporated into automated oligonucleotide synthesis by standardprocedures (H. Koster et al., Nucleic Acids Res. 12, 2261 (1984)). Thisglycine modified thyroidine analogue building block for chemical DNAsynthesis can be used to substitute one or more of the thymidine/uridinenucleotides in the nucleic acid primer sequence. The Fmoc group isremoved at the end of the solid phase synthesis with a 20 min treatmentwith a 20% solution of piperidine in DMF at room temperature. DMF isremoved by a washing step with acetonitrile and the oligonucleotidedeprotected and purified in the standard way.

EXAMPLE 7

Synthesis of a nucleic acid primer mass-modified at C-5 of theheterocyclic base of a pyrimidine nucleoside with β-alanine residues

Starting material was the same as in EXAMPLE 6. 0.281 g (1.0 mole)5-(3-Aminopropynyl-1)-2'-deoxyuridine was reacted with N-Fmoc-β-alaninepentafluorophenylester (0.955 g, 2.0 mmole) in 5 mlN,N-dimethylformamide (DMF) in the presence of 0.129 g (174 ul; 1.0mmole) N,N-disopropylethylamine for 60 min at room temperature. Solventswere removed and the product purified by silica gel chromatography asdescribed in EXAMPLE 6. Yield was 0.425 g (0.74 mmole, 74%). Anotherβ-alanine moiety can be added in exactly the same way after removal ofthe Fmoc group. The preparation of the 5'-O-dimethoxytritylatednucleoside-3'-O-β-cyanoethyl-N,N-diisopropylphosphoamidite from5-(3-(N-Fmoc-β-alanyl)-amidopropynyl-1)-2'-deoxyuridine andincorporation into automated oligonucleotide synthesis is performedunder standard conditions. This building block can substitute for any ofthe thymidine/uridine residues in the nucleic acid primer sequence. Inthe case of only one incorporated mass-modified nucleotide, the nucleicacid primer molecules prepared according to EXAMPLES 6 and 7 would havea mass difference of 14 daltons.

EXAMPLE 8

Synthesis of a nucleic acid primer mass-modified at C-5 of theheterocyclic base of a pyrimidine nucleoside with ethylene glycolmonomethyl ether

As a nucleoside component, 5-(3-aminopropynyl-1)-2'-deoxyuridine wasused in this example (see EXAMPLES 6 and 7). The mass-modifyingfunctionality was obtained as follows: 7.61 g (100.0 mmole) freshlydistilled ethylene glycol monomethyl ether dissolved in 50 ml absolutepyridine was reacted with 10.01 g (100.0 mmole) recrystallized succinicanhydride in the presence of 1.22 g (10.0 mmole)4-N,N-dimethylaminopyridine overnight at room temperature. The reactionwas terminated by the addition of water (5.0 ml), the reaction mixtureevaporated in vacuo, co-evaporated twice with dry toluene (20 ml each)and the residue redissolved in 100 ml dichloromethane. The solution wasextracted successively, twice with 10% aqueous citric acid (2×20 ml) andonce with water (20 ml) and the organic phase dried over anhydroussodium sulfate. The organic phase was evaporated in vacuo, the residueredissolved in 50 ml dichloromethane and precipitated into 500 mlpentane and the precipitate dried in vacuo. Yield was 13.12 g (74.0mmole; 74%). 8.86 g (50.0 mmole) of succinylated ethylene glycolmonomethyl ether was dissolved in 100 ml dioxane containing 5% drypyridine (5 ml) and 6.96 g (50.0 mmole) 4-nitrophenol and 10.32 g (50.0mmole) dicyclohexylcarbodiimide was added and the reaction run at roomtemperature for 4 hours. Dicyclohexylurea was removed by filtration, thefiltrate evaporated in vacuo and the residue redissolved in 50 mlanhydrous DMF. 12.5 ml (about 12.5 mmole 4-nitrophenylester) of thissolution was used to dissolve 2.81 g (10.0 mmole)5-(3-aminopropynyl-1)-2'-deoxyuridine. The reaction was performed in thepresence of 1.01 g (10.0 mmole; 1.4 ml) triethylamine at roomtemperature overnight. The reaction mixture was evaporated in vacuo,co-evaporated with toluene, redissolved in dichloromethane andchromatographed on silica gel (Si60, Merck; column 4×50 cm) withdichloromethane/methanol mixtures. The fractions containing the desiredcompound were collected, evaporated, redissolved in 25 mldichloromethane and precipitated into 250 ml pentane. The driedprecipitate of 5-(3-N-(O-succinyl ethylene glycol monomethylether)-amidopropynyl-1)-2'-deoxyuridine (yield: 65%) is5'-O-dimethoxytritylated and transformed into thenucleoside-3'-O-β-cyanoethyl-N,N-diisopropylphosphoamidite andincorporated as a building block in the automated oligonucleotidesynthesis according to standard procedures. The mass-modified nucleotidecan substitute for one or more of the thymidine/uridine residues in thenucleic acid primer sequence. Deprotection and purification of theprimer oligonucleotide also follows standard procedures.

EXAMPLE 9

Synthesis of a nucleic acid primer mass-modified at C-5 of theheterocyclic base of a pyrimidine nucleoside with diethylene glycolmonomethyl ether

Nucleoside starting material was as in previous examples,5-(3-aminopropynyl-1)-2'-deoxyuridine. The mass-modifying functionalitywas obtained similar to EXAMPLE 8. 12.02 g (100.0 mmole) freshlydistilled diethylene glycol monomethyl ether dissolved in 50 ml absolutepyridine was reacted with 10.01 g (100.0 mmole) recrystallized succinicanhydride in the presence of 1.22 g (10.0 mmole)4-N,N-dimethylaminopyridine (DMAP) overnight at room temperature. Thework-up was as described in EXAMPLE 8. Yield was 18.35 g (82.3 mmole,82.3%). 11.06 g (50.0 mmole) of succinylated diethylene glycolmonomethyl ether was transformed into the 4-nitrophenylester and,subsequently, 12.5 mmole was reacted with 2.81 g (10.0 mmole) of5-(3-aminopropynyl-1)-2'-deoxyuridine as described in EXAMPLE 8. Yieldafter silica gel column chromatography and precipitation into pentanewas 3.34 g (6.9 mmole, 69%). After dimethoxytritylation andtransformation into the nucleoside-β-cyanoethylphosphoamidite, themass-modified building block is incorporated into automated chemical DNAsynthesis according to standard procedures. Within the sequence of thenucleic acid primer UP^(0-i), one or more of the thymidine/uridineresidues can be substituted by this mass-modified nucleotide. In thecase of only one incorporated mass-modified nucleotide, the nucleic acidprimers of EXAMPLES 8 and 9 would have a mass difference of 44.05daltons.

EXAMPLE 10

Synthesis of a nucleic acid primer mass-modified at C-8 of theheterocyclic base of deoxyadenosine with glycine

Starting material was N⁶-benzoyl-8-bromo-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine preparedaccording to literature (Singh et al., Nucleic Acids Res. 18, 3339-45(1990)). 632.5 mg (1.0 mmole) of this 8-bromo-deoxyadenosine derivativewas suspended in 5 ml absolute ethanol and reacted with 251.2 mg (2.0mmole) glycine methyl ester (hydrochloride) in the presence of 241.4 mg(2.1 mmole; 366 ul) N,N-diisopropylethylamine and refluxed until thestarting nucleoside material had disappeared (4-6 hours) as checked bythin layer chromatography (TLC). The solvent was evaporated and theresidue purified by silica gel chromatography (column 2.5×50 cm) usingsolvent mixtures of chloroform/methanol containing 0.1% pyridine. Theproduct fractions were combined, the solvent evaporated, the fractionsdissolved in 5 ml dichloromethane and precipitated into 100 ml pentane.Yield was 487 mg (0.76 mmole, 76%). Transformation into thecorresponding nucleoside-β-cyanoethylphosphoamidite and integration intoautomated chemical DNA synthesis is performed under standard conditions.During final deprotection with aqueous concentrated ammonia, the methylgroup is removed from the glycine moiety. The mass-modified buildingblock can substitute one or more deoxyadenosine/adenosine residues inthe nucleic acid primer sequence.

EXAMPLE 11

Synthesis of a nucleic acid primer mass-modified at C-8 of theheterocyclic base of deoxyadenosine with glycylglycine

This derivative was prepared in analogy to the glycine derivative ofEXAMPLE 10. 632.5 mg (1.0 mmole) N⁶-Benzoyl-8-bromo-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine wassuspended in 5 ml absolute ethanol and reacted with 324.3 mg (2.0 mmole)glycyl-glycine methyl ester in the presence of 241.4 mg (2.1 mmole, 366μl) N,N-diisopropylethylamine. The mixture was refluxed and completenessof the reaction checked by TLC. Work-up and purification was similar tothat described in EXAMPLE 10. Yield after silica gel columnchromatography and precipitation into pentane was 464 mg (0.65 mmole,65%). Transformation into the nucleoside-β-cyanoethylphosphoamidite andinto synthetic oligonucleotides is done according to standardprocedures. In the case where only one of the deoxyadenosine/adenosineresidues in the nucleic acid primer is substituted by this mass-modifiednucleotide, the mass difference between the nucleic acid primers ofEXAMPLES 10 and 11 is 57.03 daltons.

EXAMPLE 12

Synthesis of a nucleic acid primer mass-modified at the C-2' of thesugar moiety of 2'-amino-2'-deoxythymidine with ethylene glycolmonomethyl ether residues

Starting material was5'-O-(4,4-dimethoxytrityl)-2'-amino-2'-deoxythymidine synthesizedaccording to published procedures (e.g., Verheyden et al., J. Org. Chem.36, 250-254 (1971); Sasaki et al., J. Org. Chem. 41, 3138-3143 (1976);Imazawa eta al., J. Org. Chem. 44, 2039-2041 (1979); Hobbs et al., J.Org. Chem. 42, 714-719 (1976); Ikehara et al., Chem. Pharm. Bull. Japan26, 240-244 (1978); see also PCT Application WO 88/00201).5'-O-(4,4-Dimethoxytrityl)-2'-amino-2'-deoxythymidine (559.62 mg; 1.0mmole) was reacted with 2.0 mmole of the 4-nitrophenyl ester ofsuccinylated ethylene glycol monomethyl ether (sec EXAMPLE 8) in 10 mldry DMF in the presence of 1.0 mmole (140 μl) triethylamine for 18 hoursat room temperature. The reaction mixture was evaporated in vacuo,co-evaporated with toluene, redissolved in dichloromethane and purifiedby silica gel chromatography (Si60, Merck; column: 2.5×50 cm; eluent:chloroform/methanol mixtures containing 0.1% triethylamine). The productcontaining fractions were combined, evaporated and precipitated intopentane. Yield was 524 mg (0.73 mmol; 73%). Transformation into thenucleoside-β-cyanoethyl-N,N-diisopropylphosphoamidite and incorporationinto the automated chemical DNA synthesis protocol is performed bystandard procedures. The mass-modified deoxythymidine derivative cansubstitute for one or more of the thymidine residues in the nucleic acidprimer.

In an analogous way, by employing the 4-nitrophenyl ester ofsuccinylated diethylene glycol monomethyl ether (see EXAMPLE 9) andtriethylene glycol monomethyl ether, the corresponding mass-modifiedoligonucleotides are prepared. In the case of only one incorporatedmass-modified nucleoside within the sequence, the mass differencebetween the ethylene, diethylene and triethylene glycol derivatives is44.05, 88.1 and 132.15 daltons respectively.

EXAMPLE 13

Synthesis of a nucleic acid primer mass-modified in the internucleotidiclinkage via alkylation of phosphorothioate groups

Phosphorothioate-containing oligonucleotides were prepared according tostandard procedures (see e.g. Gait et al., Nucleic Acids Res., 19 1183(1991)). One, several or all internucleotide linkages can be modified inthis way. The (-)-M13 nucleic acid palmer sequence (17-mer)5'-dGTAAAACGACGGCCAGT was synthesized in 0.25 μmole scale on a DNAsynthesizer and one phosphorothioate group introduced after the finalsynthesis cycle (G to T coupling). Sulfurization, deprotection andpurification followed standard protocols. Yield was 31.4 nmole (12.6%overall yield), corresponding to 31.4 nmole phosphorothioate groups.Alkylation was performed by dissolving the residue in 31.4 μl TE buffer(0.01M Tris pH 8.0, 0.001M EDTA) and by adding 16 μl of a solution of 20mM solution of 2-iodoethanol (320 nmole; i.e., 10-fold excess withrespect to phosphorothioate diesters) in N,N-dimethylformamide (DMF).The alkylated oligonucleotide was purified by standard reversed phaseHPLC (RP-18 Ultraphere, Beckman; column: 4.5×250 mm; 100 mMtriethylammonium acetate, pH 7.0 and a gradient of 5 to 40%acetonitrile).

In a variation of this procedure, the nucleic acid primer containing oneor more phosphorothioate phosphodiester bond is used in the Sangersequencing reactions. The primer-extension products of the foursequencing reactions are purified as exemplified in EXAMPLES 1-4,cleaved off the solid support, lyophilized and dissolved in 4 μl each ofTE buffer pH 8.0 and alkylated by addition of 2 μl of a 20 mM solutionof 2-iodoethanol in DMF. It is then analyzed by ES and/or MALDI massspectrometry.

In an analogous way, employing instead of 2-iodoethanol, e.g.,3-iodopropanol, 4-iodobutanol mass-modified nucleic acid primer areobtained with a mass difference of 14.03, 28.06 and 42.03 daltonsrespectively compared to the unmodified phosphorothioatephosphodiester-containing oligonucleotide.

EXAMPLE 14

Synthesis of 2'-amino-2'-deoxyuridine-5'-triphosphate and3'-amino-2',3'-dideoxythymidine-5'-triphosphate mass-modified at the 2'-or 3'-amino function with glycine or β-alanine residues

Starting material was 2'-azido-2'-deoxyuridine prepared according toliterature (Verheyden et al. J. Org. Chem. 36, 250 (1971)), which was4,4-dimethoxytritylated at 5'-OH with 4,4-dimethoxytrityl chloride inpyridine and acetylated at 3'-OH with acetic anhydride in a one-potreaction using standard reaction conditions. With 191 mg (0.71 mmole)2'-azido-2'-deoxyuridine as starting material, 396 mg (0.65 mmol, 90.8%)5'-O-(4,4-dimethoxytrityl)-3'-O-acetyl-2'-azido-2'-deoxuridine wasobtained after purification via silica gel chromatography. Reduction ofthe azido group was performed using published conditions (Barta et al.,Tetrahedron 46, 587-594 (1990)). Yield of5'-O-(4,4-dimethoxytrityl)-3'-O-acetyl-2'-amino-2'-deoxyuridine aftersilica gel chromatography was 288 mg (0.49 mmole; 76%). This protected2'-amino-2'-deoxyuridine derivative (588 mg, 1.0 mmole) was reacted with2 equivalents (927 mg, 2.0 mmole) N-Fmoc-glycine pentafluorophenyl esterin 10 ml dry DMF overnight at room temperature in the presence of 1.0mmole (174 μl) N,N-diisopropylethylamine. Solvents were removed byevaporation in vacuo and the residue purified by silica gelchromatography. Yield was 711 mg (0.71 mmole, 82%). Detritylation wasachieved by a one hour treatment with 80% aqueous acetic acid at roomtemperature. The residue was evaporated to dryness, co-evaporated twicewith toluene, suspended in 1 ml dry acetonitrile and 5'-phosphorylatedwith POCl₃ according to literature (Yoshikawa et al., Bull. Chem. Soc.Japan 42, 3505 (1969) and Sowa et al., Bull. Chem. Soc. Japan 48, 2084(1975)) and directly transformed in a one-pot reaction to the5'-triphosphate using 3 ml of a 0.5M solution (1.5 mmole) tetra(tri-n-butylammonium)pyrophosphate in DMF according to literature (e.g.Seela et al., Helvetica Chimica Acta 74, 1048 (1991)). The Fmoc and the3'-O-acetyl groups were removed by a one-hour treatment withconcentrated aqueous ammonia at room temperature and the reactionmixture evaporated and lyophilized. Purification also followed standardprocedures by using anion-exchange chromatography on DEAE-Sephadex witha linear gradient of triethylammonium bicarbonate (0.1M-1.0M).Triphosphate containing fractions (checked by thin layer chromatographyon polyethyleneimine cellulose plates) were collected, evaporated andlyophilized. Yield (by UV-absorbance of the uracil moiety) was 68% (0.48mmole).

A glycyl-glycine modified 2'-amino-2'-deoxyuridine-5'-triphosphate wasobtained by removing the Fmoc group from5'-O-(4,4-dimethoxytrityl)-3'-O-acetyl-2'-N-(N-9-fluorenylmethyloxycarbonyl-glycyl)-2'-amino-2'-deoxyuridineby a one-hour treatment with a 20% solution of piperidine in DMF at roomtemperature, evaporation of solvents, two-fold co-evaporation withtoluene and subsequent condensation with N-Fmoc-glycinepentafluorophenyl ester. Starting with 1.0 mmole of the2'-N-glycyl-2'-amino-2'-deoxyuridine derivative and following theprocedure described above, 0.72 mmole (72%) of the corresponding2'-(N-glycyl-glycyl)-2'-amino-2'-deoxyuridine-5'-triphosphate wasobtained.

Starting with5'-O-(4,4-dimethoxytrityl)-3'-O-acetyl-2'-amino-2'-deoxyuridine andcoupling with N-Fmoc-β-alanine pentafluorophenyl ester, thecorresponding 2'-(N-β-alanyl)-2'-amino-2'-deoxyuridine-5'-triphosphatecan be synthesized. These modified nucleoside triphosphates areincorporated during the Sanger DNA sequencing process in theprimer-extension produces. The mass difference between the glycine,β-alanine and glycyl-glycine mass-modified nucleosides is, pernucleotide incorporated, 58.06, 72.09 and 115.1 daltons respectively.

When starting with5'-O-(4,4-dimethoxytrityl)-3'-amino-2',3'-dideoxythymidine (obtained bypublished procedures, see EXAMPLE 12), the corresponding3'-(N-glycyl)-3'-amino-/3'-(-N-glycyl-glycyl)-3'-amino-/and3'-(N-β-alanyl)-3'-amino-2',3'-dideoxythymidine-5'-triphosphates can beobtained. These mass-modified nucleoside triphosphates serve as aterminating nucleotide unit in the Sanger DNA sequencing reactionsproviding a mass difference per terminated fragment of 58.06, 72.09 and115.1 daltons respectively when used in the multiplexing sequencingmode. The mass-differentiated fragments can then be analyzed by ESand/or MALDI mass spectrometry.

EXAMPLE 15

Synthesis of deoxyuridine-5'-triphosphate mass-modified at C-5 of theheterocyclic base with glycine, glycyl-glycine and β-alanine residues

0.281 g (1.0 mmole) 5-(3-Aminopropynyl-1)-2'-deoxyuridine (see EXAMPLE6) was reacted with either 0.927 g (2.0 mmole) N-Fmoc-glycinepentafluorophenylester or 0.955 g (2.0 mmol) N-Fmoc-β-alaninepentafluorophenyl ester in 5 ml dry DMF in the presence of 0.129 gN,N-diisopropylethylamine (174 ul, 1.0 mmole) overnight at roomtemperature. Solvents were removed by evaporation in vacuo and thecondensation products purified by flash chromatography on silica gel(Still et al., J. Org. Chem, 43, 2923-2925 (1978)). Yields were 476 mg(0.85 mmole: 85%) for the glycine and 436 mg (0.76 mmole; 76%) for theβ-alanine derivatives. For the synthesis of the glycyl-glycinederivative, the Fmoc group of 1.0 mmole Fmoc-glycine-deoxyuridinederivative was removed by one-hour treatment with 20% piperidine in DMFat room temperature. Solvents were removed by evaporation in vacuo, theresidue was co-evaporated twice with toluene and condensed with 0.927 g(2.0 mmole) N-Fmoc-glycine pentafluorophenyl ester and purified asdescribed above. Yield was 445 mg (0.72 mmole; 72%). The glycyl-,glycyl-glycyl- and β-alanyl-2'-deoxyuridine derivatives, N-protectedwith the Fmoc group were transformed to the 3'-O-acetyl derivatives bytritylation with 4,4-dimethoxytrityl chloride in pyridine andacetylation with acetic anhydride in pyridine in a one-pot reaction andsubsequently detritylated by one hour treatment with 80% aqueous aceticacid according to standard procedures. Solvents were removed, theresidues dissolved in 100 ml chloroform and extracted twice with 50 ml10% sodium bicarbonate and once with 50 ml water, dried with sodiumsulfate, the solvent evaporated and the residues purified by flashchromatography on silica gel. Yields were 361 mg (0.60 mmole; 71%) forthe glycyl-, 351 mg (0.57 mmole; 75%) for the β-alanyl- and 323 mg (0.49mmole; 68%) for the glycyl-glycyl-3-O'-acetyl-2'-deoxyuridinederivatives respectively. Phosphorylation at the 5'-OH with POCl₃,transformation into the 5'-triphosphate by in-situ reaction withtetra(tri-n-butylammonium)pyrophosphate in DMF, 3'-de-O-acetylation,cleavage of the Fmoc group, and final purification by anion-exchangechromatography on DEAE-Sephadex was performed as described in EXAMPLE14. Yields according to UV-absorbance of the uracil moiety were 0.41mmole 5-(3-(N-glycyl)-amidopropynyl-1)-2'-deoxyuridine-5'-triphosphate(84%), 0.43 mmole5-(3-(N-β-alanyl)-amidopropynyl-1)-2'-deoxyuridine-5'-triphosphate (75%)and 0.38 mmole5-(3-(N-glycyl-glycyl)-amidopropynyl-1)-2'-deoxyuridine-5'-triphosphate(78%).

These mass-modified nucleoside triphosphates were incorporated duringthe Sanger DNA sequencing primer-extension reactions.

When using 5-(3-aminopropynyl-1)-2',3'-dideoxyuridine as startingmaterial and following an analogous reaction sequence the correspondingglycyl-, glycyl-glycyl- andβ-alanyl-2',3'-dideoxyuridine-5'-triphosphates were obtained in yieldsof 69, 63 and 71% respectively. These mass-modified nucleosidetriphosphates serve as chain-terminating nucleotides during the SangerDNA sequencing reactions. The mass-modified sequencing ladders areanalyzed by either ES or MALDI mass spectrometry.

EXAMPLE 16

Synthesis of 8-glycyl- and8-glycyl-glycyl-2'-deoxyadenosine-5'-triphosphate

727 mg (1.0 mmole) ofN6-(4-tert-butylphenoxyacetyl)-8-glycyl-5'-(4,4-dimethoxytrityl)-2'-deoxyadenosineor 800 mg (1.0 mmole) N⁶-(4-tert-butylphenoxyacetyl)-8-glycyl-glycyl-5'-(4,4-dimethoxytrityl)-2'-deoxyadenosineprepared according to EXAMPLES 10 and 11 and literature (Koster et al.,Tetrahedron 37, 362 (1981)) were acetylated with acetic anhydride inpyridine at the 3'-OH, detritylated at the 5'-position with 80% aceticacid in a one-pot reaction and transformed into the 5'-triphosphates viaphosphorylation with POCl₃ and reaction in-situ withtetra(tri-n-butylammonium)pyrophosphate as described in EXAMPLE 14.Deprotection of the N⁶ -tert-butylphenoxyacetyl, the 3'-O-acetyl and theO-methyl group at the glycine residues was achieved with concentratedaqueous ammonia for ninety minutes at room temperature. Ammonia wasremoved by lyophilization and the residue washed with dichloromethane,solvent removed by evaporation in vacuo and the remaining solid materialpurified by anion-exchange chromatography on DEAE-Sephadex using alinear gradient of triethylammonium bicarbonate from 0.1 to 1.0M. Thenucleoside triphosphate containing fractions (checked by TLC onpolyethyleneimine cellulose plates) were combined and lyophillized.Yield of the 8-glycyl-2'-deoxyadenosine-5'-triphosphate (determined byUV-absorbance of the adenine moiety) was 57% (0.57 mmole). The yield forthe 8-glycyl-glycyl-2'-deoxyadenosine-5'-triphosphate was 51% (0.51mmole).

These mass-modified nucleoside triphosphates were incorporated duringprimer-extension in the Sanger DNA sequencing reactions.

When using the corresponding N6-(4-tert-butylphenoxyacetyl)-8-glycyl- or-glycyl-glycyl-5'-O-(4,4-dimethoxytrityl)-2',3'-dideoxyadenosinederivatives as starting materials prepared according to standardprocedures (see, e.g., for the introduction of the 2',3'-function: Seelaet al., Helvetica Chimica Acta 74, 1048-1058 (1991)) and using ananalogous reaction sequence as described above, the chain-terminatingmass-modified nucleoside triphosphates 8-glycyl- and8-glycyl-glycyl-2'.3'-dideoxyadenosine-5'-triphosphates were obtained in53 and 47% yields respectively. The mass-modified sequencing fragmentladders are analyzed by either ES or MALDI mass spectrometry.

EXAMPLE 17

Mass-modification of Sanger DNA sequencing fragment ladders byincorporation of chain-elongating 2'-deoxy- and chain-terminating2',3'-dideoxythymidine-5'-(alpha-S-)-triphosphate and subsequentalkylation with 2-iodoethanol and 3-iodopropanol

2',3'-Dideoxythymidine-5'-(alpha-S)-triphosphate was prepared accordingto published procedures (e.g., for the alpha-S-triphosphate moiety:Eckstein et al., Biochemistry 15, 1685 (1976) and Accounts Chem. Res,12, 204 (1978) and for the 2',3'-dideoxy moiety: Seela et al., HelveticaChimica Acta, 74, 1048-1058 (1991)). Sanger DNA sequencing reactionsemploying 2'-deoxythymidine-5'-(alpha-S)-triphosphate are performedaccording to standard protocols (e.g. Eckstein, Ann. Rev. Biochem, 54,367 (1985)). When using2',3'-dideoxythymidine-5'-(alpha-S)-triphosphates, this is used insteadof the unmodified 2',3'-dideoxythymidine-5'-triphosphate in standardSanger DNA sequencing (see e.g. Swerdlow et al., Nucleic Acids Res. 18,1415-1419 (1990)). The template (2 pmole) and the nucleic acid M13sequencing primer (4 pmole) modified according to EXAMPLE 1 are annealedby heating to 65° C. in 100 ul of 10 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 50mM NaCl, 7 mM dithiothreitol (DTT) for 5 min and slowly brought to 37°C. during a one hour period. The sequencing reaction mixtures contain,as exemplified for the T-specific termination reaction, in a finalvolume of 150 ul, 200 uM (final concentration) each of dATP, dCTP, dTTP,300 uM c7-deaza-dGTP, 5 uM2',3'-dideoxythymidine-5'-(alpha-S)-triphosphate and 40 units Sequenase(United States Biochemicals). Polymerization is performed for 10 min at37° C., the reaction mixture heated to 70° C. to inactivate theSequenase, ethanol precipitated and coupled to thiolated Sequelonmembrane disks (8 mm diameter) as described in EXAMPLE 1. Alkylation isperformed by treating the disks with 10 ul of 10 mM solution of either2-iodoethanol or 3-iodopropanol in NMM(N-methylmorpholine/water/2-propanol, 2/49/49, v/v/v) (three times),washing with 10 ul NMM (three times) and cleaving the alkylatedT-terminated primer-extension products off the support by treatment withDTT as described in EXAMPLE 1. Analysis of the mass-modified fragmentfamilies is performed with either ES or MALDI mass spectrometry.

EXAMPLE 18

Analysis of a Mixture of Oligothymidylic Acids

Oligothymidylic acid, oligo p(dT)₁₂₋₁₈, is commercially available(United States Biochemical, Cleveland, Ohio). Generally, a matrixsolution of 0.5M in ethanol was prepared. Various matrices were used forthis Example and Examples 19-21 such as 3,5-dihydroxybenzoic acid,sinapinic acid, 3-hydroxypicolinic acid, 2,4,6-trihydroxyacetophenone.Oligonucleotides were lyophilized after purification by HPLC and takenup in ultrapure water (MilliQ, Millipore) using amounts to obtain aconcentration of 10 pmoles/μl as stock solution. An aliquot (1 μl) ofthis concentration or a dilution in ultrapure water was mixed with 1 μlof the matrix solution on a flat metal surface serving as the probe tipand dried with a fan using cold air. In some experiments, cation-ionexchange beads in the acid form were added to the mixture of matrix andsample solution.

MALDI-TOF spectra were obtained for this Example and Examples 19-21 ondifferent commercial instruments such as Vision 2000 (Finnigan-MAT), VGTofSpcc (Fisons Instruments), LaserTec Research (Vestec). The conditionsfor this Example were linear negative ion mode with an accelerationvoltage of 25 kV. The MALDI-TOF spectrum generated is shown in FIG. 14.Mass calibration was done externally and generally achieved by usingdefined peptides of appropriate mass range such as insulin, gramicidinS, trypsinogen, bovine serum albumen, and cytochrome C. All spectra weregenerated by employing a nitrogen laser with 5 nsec pulses at awavelength of 337 nm. Laser energy varied between 10⁶ and 10⁷ W/cm². Toimprove signal-to-noise ratio generally, the intensities of 10 to 30laser shots were accumulated.

EXAMPLE 19

Mass Spectrometric Analysis of a 50-mer and a 99-mer

Two large oligonucleotides were analyzed by mass spectrometry. The50-mer d(TAACGGTCATTACGGCCATTGACTGTAGGACCTGCATTACATGACTAGCT) (SEQ IDNO:3) and dT(pdT)₉₉ were used. The oligodeoxynueleotides weresynthesized using β-cyanoethylphosphoamidites and purified usingpublished procedures. (e.g. N. D. Sinha, J. Biernat, J. McManus and H.Koster, Nucleic Acids Res., 12, 4539 (1984)) employing commerciallyavailable DNA synthesizers from either Millipore (Bedford, Mass.) orApplied Biosystems (Foster City, Calif.) and HPLC equipment and RP 18reverse phase columns from Waters (Milford, Mass.). The samples for massspectrometric analysis were prepared as described in Example 18. Theconditions used for MALDI-MS analysis of each oligonucleotide were 500fmol of each oligonucleotide, reflection positive ion mode with anacceleration of 5 kV and postacceleration of 20 kV. The MALDI-TOFspectra generated were superimposed and are shown in FIG. 15.

EXAMPLE 20

Simulation of the DNA Sequencing Results of FIG. 2

The 13 DNA sequences representing the nested dT-terminated fragments ofthe Sanger DNA sequencing for the 50-mer described in Example 19 (SEQ IDNO:3) were synthesized as described in Example 19. The samples weretreated and 500 fmol of each fragment was analyzed by MALDI-MS asdescribed in Example 18. The resulting MALDI-TOF spectra are shown inFIG. 16. The conditions were reflectron positive ion mode with anacceleration of 5 kV and postacceleration of 20 kV. Calculated molecularmasses and experimental molecular masses are shown in Table 1.

The MALDI-TOF spectra were superimposed (FIG. 17) to demonstrate thatthe individual peaks are resolvable even between the 10-mer and 11-mer(upper panel) and the 37-mer and 38-mer (lower panel). The two panelsshow two different scales and the spectra analyzed at that scale.

EXAMPLE 21

MALDI-MS Analysis of a Mass-Modified Oligonucleotide

A 17-mer was mass-modified at C-5 of one or two deoxyuridine moieties.5-13-(2-Methoxyethoxyl)-tridecyne-1-yl!-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyuridine-3'-β-cyanoethyl-N-N-diisopropylphosphoamiditewas used to synthesize the modified 17-mers using the methods describedin Example 19.

The modified 17-mers were ##STR1##

The samples were prepared and 500 fmol of each modified 17-mer wasanalyzed using MALDI-MS as described in Example 18. The conditions usedwere reflectron positive ion mode with an acceleration of 5 kV andpostacceleration of 20 kV. The MALDI-TOF spectra which were generatedwere superimposed and are shown in FIG. 18.

All of the above-cited references and publications are herebyincorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of this invention and are covered by the followingclaims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CATGCCATGGCATG14                                                              (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AAATTGTGCACATCCTGCAGC21                                                       (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TAACGGTCATTACGGCCATTGACTGTAGGACCTGCATTACATGACTAGCT50                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TAAAACGACGGGCCAGXG18                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       XAAAACGACGGGCCAGXG18                                                          __________________________________________________________________________

I claim:
 1. A kit for sequencing one or more species of nucleic acids bymultiplex mass spectrometric nucleic acid sequencing, comprising:a) asolid support having a linking functionality (L'); b) a set of nucleicacid primers suitable for initiating synthesis of a set of nucleic acidswhich are complementary to the different species of nucleic acids, theprimers each including a linking group (L) able to interact with thelinking functionality (L') and reversibly link the primers to the solidsupport and optionally, a tag probe; c) a set of chain-elongatingnucleotides for synthesizing the complementary nucleic acids; d) a setof chain-terminating nucleotides for terminating synthesis of thecomplementary nucleic acids and generating sets of base-specificterminated complementary nucleic acid fragments; and e) a polymerase forsynthesizing the complementary nucleic acids from the nucleic acidprimers, chain-elongating nucleotides and terminatingnucleotides,wherein in the absence of a tag probe, at least one reagentselected from the group consisting of the primers, the chain-elongatingnucleotides, and the chain-terminating nucleotides is mass modified toprovide distinction between each set of base-specifically terminatednucleotides of each species of nucleic acid by mass spectrometry.
 2. Akit of claim 1, wherein the set of chain-elongating nucleotides iscomprised of a nucleotide selected from the group consisting ofdeoxyadenosine triphosphate (dATP) deoxythymidine triphosphate (dTTP),deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP),deoxyinosine triphosphate (dITP), 7-deaza deoxyadenosine triphosphate(c⁷ dATP), 7-deaza deoxythymidine triphosphate (C⁷ TTP), 7-deazadeoxyguanosine triphosphate (c⁷ dGTP), 7-deaza deoxycytidinetriphosphate (c⁷ dCTP) and 7-deaza deoxyinosine triphosphate (c⁷ dITP).3. A kit of claim 1, wherein the set of chain-terminating nucleotides iscomprised of the following nucleotides: dideoxyadenosine triphosphate(ddATP), dideoxythymidine triphosphate (ddTTP), dideoxyguanosinetriphosphate (ddGTP), dideoxycytidine triphosphate (ddCTP), 7-deazadideoxyguanosine triphosphate (c⁷ ddGTP), 7-deaza dideoxyadenosinetriphosphate (c⁷ ddATP), 7-deaza dideoxyinosine triphosphate (c⁷ ddITP).4. A kit of claim 1, wherein the set of chain elongating nucleotides iscomprised of a nucleotide selected from the group consisting ofadenosine triphosphate (ATP), uridine triphosphate (UTP), guanosinetriphosphate (GTP), cytidine triphosphate (CTP), inosine triphosphate(ITP), 7-deaza adenosine triphosphate (c⁷ ATP), 7-deaza guanosinetriphosphate (c⁷ GTP), and 7-deaza inosine triphosphate (c⁷ ITP).
 5. Akit of claim 1, wherein the set of chain elongating nucleotides iscomprised of the following nucleotides: deoxyadenosine triphosphate(3'-dATP), deoxyuridine triphosphate (3'-dUTP), deoxyguanosinetriphosphate (3'-dGTP), deoxycytidine triphosphate (3'-dCTP), 7-deaza3'deoxyadenosine triphosphate (c⁷ -3'dATP), 7-deaza 3'deoxyguanosinetriphosphate (c⁷ -3'dGTP) and 7-deaza 3'deoxy o!inosine (c⁷ -3'dITP). 6.A kit of claim 1, wherein the linkage between the linking group (L) andthe linking functionality (L') is selected from the group consisting of:a photocleavable bond, a tritylether bond, a β-benzoylpropionyl group, alevulinyl group, a disulfide bond, an arginine/arginine bond, alysine/lysine bond, and a pyrophosphate bond and a bond created byWatson-Crick base pairing.
 7. A kit of claim 1, wherein the massmodified reagent is modified with a mass-modifying functionality (M) ata heterocyclic base of at least one nucleotide.
 8. The kit of claim 7,wherein the heterocyclic base-modified nucleotide is selected from thegroup consisting of a cytosine nucleotide modified at C-5, a thyminenucleotide modified at C-5, a thymine nucleotide modified at the C-5methyl group, a uracil nucleotide modified at C-5, an adenine nucleotidemodified at C-8, an adenine nucleotide modified at C-7, a c⁷-deazaadenine modified at C-8, a c⁷ -deazaadenine modified at C-7, aguanine nucleotide modified at C-8, a guanine nucleotide modified atC-7, a c⁷ -deazaguanine modified at C-8, a c⁷ -deazaguanine modified atC-7, a hypoxanthine modified at C-8, a c⁷ -deazahypoxanthine modified atC-7, and a c⁷ -deazahypoxanthine modified at C-8.
 9. A kit of claim 1,wherein the mass modified reagent is modified with a mass-modifyingfunctionality (M) attached to one or more phosphate moieties of theinternucleotidic linkages of the fragments.
 10. A kit of claim 1,wherein the mass modified reagent is modified with a mass-modifyingfunctionality (M) attached to one or more sugar moieties of nucleotideswithin the set of mass modified base-specifically terminated fragmentsat at least one sugar position selected from the group consisting of aC-2' position, an external C-3' position, and an external C-5' position.11. A kit of claim 1, wherein the mass modified reagent is modified witha mass-modifying functionality (M) attached to the sugar moiety of a5'-terminal nucleotide and wherein the mass-modifying function (M) isthe linking functionality (L).
 12. A kit of claim 1, wherein thebase-specifically terminated nucleic acid fragments are generated usingat least one reagent selected from the group consisting of a nucleicacid primer, a chain-elongating nucleotide, a chain-terminatingnucleotide and a tag probe which has been modified with a precursor ofthe mass-modifying functionality, M; and a subsequent step comprisesmodifying the precursor of the mass-modifying functionality, M, togenerate the mass-modifying functionality, M, prior to massspectrometric analysis.
 13. A kit of claim 1, wherein the primer or thetag probe is comprised of a deoxyribonucleotide selected from the groupconsisting of: a 7-deaza deoxyadenosine triphosphate, (c⁷ dA), a 7-deazadeoxyguanosine triphosphate (c⁷ dG) and a 7-deaza deoxyinosinetriphosphate (c⁷ dI).
 14. A kit of claim 1, wherein the primer or thetag probe is comprised of a ribonucleotide selected from the groupconsisting of: 7-deaza adenine (c⁷ A), 7-deaza guanine (c⁷ G) and7-deaza inosine (c⁷ I).
 15. A kit for sequencing nucleic acids by massspectrometry, comprising:a) a solid support having a linkingfunctionality (L'); b) a set of nucleic acid primers suitable forinitiating synthesis of a set of complementary nucleic acids which arecomplementary to the different species of nucleic acids, the primerseach including a linking group (L) able to interact with the linkingfunctionality (L') and reversibly immobilize the primers on the solidsupport; c) a set of chain-elongating nucleotides for synthesizing thecomplementary nucleic acids; d) a set of chain-terminating nucleotidesfor terminating synthesis of the complementary nucleic acids andgenerating sets of base-specific terminated complementary nucleic acidfragments; and e) a polymerase for synthesizing the complementarynucleic acids from the primers, chain-elongating nucleotides andchain-terminating nucleotides,wherein the chain-terminating nucleotidesare mass-modified so that addition of one species of thechain-terminating nucleotides to the complementary nucleic acid can bedistinguished by mass spectrometry from addition of all other species ofchain-terminating nucleotides concurrently analyzed.
 16. A kit of claim15, wherein the set of chain-elongating nucleotides is comprised of anucleotide selected from the group consisting of deoxyadenosinetriphosphate (dATP) deoxythymidine triphosphate (dTTP), deoxyguanosinetriphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxyinosinetriphosphate (dITP), a 7-deazadeoxyguanosine triphosphate (c⁷ dGTP), a7-deazadeoxyadenosine triphosphate (c⁷ dATP), and a 7-deazadeoxyinosinetriphosphate (c⁷ dITP).
 17. A kit of claim 15, wherein the set ofchain-terminating nucleotides is comprised of the following nucleotides:dideoxyadenosine triphosphate (ddATP), dideoxythymidine triphosphate(ddTTP), dideoxyguanosine triphosphate (ddGTP), dideoxycytidinetriphosphate (ddCTP), 7-deazadideoxyguanosine triphosphate (c⁷ ddGTP),7-deazadideoxyadenosine triphosphate (c⁷ ddATP), 7-deazadideoxyinosinetriphosphate (c⁷ ddITP).
 18. A kit of claim 15, wherein the set of chainelongating nucleotides is comprised of a nucleotide selected from thegroup consisting of adenosine triphosphate (ATP), uridine triphosphate(UTP), guanosine triphosphate (GTP), cytidine triphosphate (CTP),inosine triphosphate (ITP), a 7-deazaadenosine triphosphate (c⁷ ATP), a7-deazaguanosine triphosphate (c⁷ GTP), and a 7-deazainosinetriphosphate (c⁷ ITP).
 19. A kit of claim 15, wherein the set of chainterminating nucleotides is comprised of the following nucleotides:deoxyadenosine triphosphate (3'-dATP), deoxyuridine triphosphate(3'-dUTP), deoxyguanosine triphosphate (3'-dGTP), deoxycytidinetriphosphate (3'-dCTP), 7-deaza 3'deoxyadenosine triphosphate (c⁷-3'dATP), 7-deaza 3'deoxyguanosine triphosphate (c⁷ -3'dGTP) and 7-deaza3'deoxy o!inosine triphosphate (c⁷ -3'dITP).
 20. A kit of claim 15,wherein the linkage between the linking group (L) and the linkingfunctionality (L') is selected from the group consisting of: aphotocleavable bond, a tritylether bond, a β-benzoylpropionyl group, alevulinyl group, a disulfide bond, an arginine/arginine bond, alysine/lysine bond, and a pyrophosphate bond and a bond created byWatson-Crick base pairing.
 21. A kit of claim 15, wherein the massmodified reagent is modified with a mass-modifying functionality (M) ata heterocyclic base of at least one nucleotide.
 22. The kit of claim 21,wherein the heterocyclic base-modified nucleotide is selected from thegroup consisting of a cytosine nucleotide modified at C-5, a thyminenucleotide modified at C-5, a thymine nucleotide modified at the C-5methyl group, a uracil nucleotide modified at C-5, an adenine nucleotidemodified at C-8, an adenine nucleotide modified at C-7, a c⁷-deazaadenine modified at C-8, a c⁷ -deazaadenine modified at C-7, aguanine nucleotide modified at C-8, a guanine nucleotide modified atC-7, a c⁷ -deazaguanine modified at C-8, a c⁷ -deazaguanine modified atC-7, a hypoxanthine modified at C-8, a c⁷ -deazahypoxanthine modified atC-7, and a c⁷ -deazahypoxanthine modified at C-8.
 23. A kit of claim 15,wherein the mass modified reagent is modified with a mass-modifyingfunctionality (M) attached to one or more phosphate moieties of theinternucleotidic linkages of the fragments.
 24. A kit of claim 15,wherein the mass modified reagent is modified with a mass-modifyingfunctionality (M) attached to one or more sugar moieties of nucleotideswithin the set of mass modified base-specifically terminated fragmentsat at least one sugar position selected from the group consisting of aC-2' position, an external C-3' position, and an external C-5' position.25. A kit of claim 15, wherein the mass modified reagent is modifiedwith a mass-modifying functionality (M) attached to the sugar moiety ofa 5'-terminal nucleotide and wherein the mass-modifying function (M) isthe linking functionality (L).
 26. A kit of claim 15, wherein thebase-specifically terminated nucleic acid fragments are generated usingat least one reagent selected from the group consisting of a nucleicacid primer, a chain-elongating nucleotide, a chain-terminatingnucleotide and a tag probe which has been modified with a precursor ofthe mass-modifying functionality, M; and a subsequent step comprisesmodifying the precursor of the mass-modifying functionality, M, togenerate the mass-modifying functionality, M, prior to massspectrometric analysis.